Colloidal metal compositions and methods

ABSTRACT

The present invention comprises compositions and methods for delivery systems of agents, including therapeutic compounds, pharmaceutical agents, drugs, detection agents, nucleic acid sequences and biological factors. In general, these vector compositions comprise a colloidal metal, derivatized PEG (polyethylene glycol) and an agent. The invention also comprises methods and compositions for making such colloidal metal compositions and for treatment of cancer.

RELATED APPLICATIONS

This application claims priority to Provisional Application Ser. No.60/287,363 filed on Apr. 30, 2001.

FIELD OF THE INVENTION

The present invention relates to compositions and methods forgeneralized delivery of agents and delivery of agents to specific sites.In general, the present invention relates to colloidal metalcompositions and methods for making and using such compositions.

BACKGROUND OF THE INVENTION

It has long been a goal of therapeutic treatment to find the magicbullet that would track to the site of need and deliver a therapeuticresponse without undue side effects. Many approaches have been tried toreach this goal. Therapeutic agents have been designed to take advantageof differences in active agents, such as hydrophobicity orhydrophilicity, or size of therapeutic particulates for differentialtreatment by cells of the body. Therapies exist that deliver therapeuticagents to specific segments of the body or to particular cells by insitu injection, and either use or overcome body defenses such as theblood-brain barrier, that limit the delivery of therapeutic agents.

One method that has been used to specifically target therapeutic agentsto specific tissues or cells is delivery based on the combination of atherapeutic agent and a binding partner of a specific receptor. Forexample, the therapeutic agent may be cytotoxic or radioactive and whencombined with a binding partner of a cellular receptor, cause cell deathor interfere with genetic control of cellular activities once bound tothe target cells. This type of delivery device requires having areceptor that is specific for the cell-type to be treated, an effectivebinding partner for the receptor, and an effective therapeutic agent.Molecular genetic manipulations have been used to overcome some of theseproblems.

Specific delivery of genetic sequences into exogenous cells or forover-expression of endogenous sequences are methods of great interest atthe current time. Various techniques for inserting genes into cells areused. These techniques include precipitation, viral vectors, directinsertion with micropipettes and gene guns, and exposure of nucleic acidto cells. A widely used precipitation technique uses calcium phosphateto precipitate DNA to form insoluble particles. The goal is for at leastsome of these particles to become internalized within the host cells bygeneralized cellular endocytosis. This results in the expression of thenew or exogenous genes. This technique has a low efficiency of entry ofexogenous genes into cells with the resulting expression of the genes.The internalization of the genes is non-specific with respect to whichcells are transfected because all exposed cells are capable ofinternalizing the exogenous genes since there is no reliance upon anyparticular recognition site for the endocytosis. This technique is usedwidely in vitro, but because of the lack of specificity of target cellselection and poor uptake by highly differentiated cells, its use invivo is not contemplated. In addition, its use in vivo is limited by theinsoluble nature of the precipitated nucleic acids.

A similar technique involves the use of DEAE-Dextran for transfectingcells in vitro. DEAE-Dextran is deleterious to cells and also results innon-specific insertion of nucleic acids into cells. This method is notadvisable in vivo.

Other techniques for transfecting cells, or providing for the entry ofexogenous genes into cells are also limited. Using viruses as vectorshas some applicability for in vitro and in vivo introduction ofexogenous genes into cells. There is always the risk that the presenceof viral proteins will produce unwanted effects in an in vivo use.Additionally, viral vectors may be limited as to the size of exogenousgenetic material that can be ferried into the cells. Repeated use ofviral vectors raises an immunological response in the recipient andlimits the times the vector can be used.

Exogenous gene delivery has also been used with liposome-entrappednucleic acids. Liposomes are membrane-enclosed sacs that can be filledwith a variety of materials, including nucleic acids. Liposome deliverydoes not provide for uniform delivery to cells because of uneven fillingof the liposomes. Though liposomes can be targeted to specific cellulartypes if binding partners for receptors are included, liposomes sufferfrom breakage problems, and thus delivery is not specific.

Brute force techniques for inserting exogenous nucleic acids includepuncturing cellular membranes with micropipettes or gene guns to insertexogenous DNA into a cell. These techniques work well for someprocedures, but are not widely applicable. They are highly laborintensive and require very skilled manipulation of the recipient cell.These are not techniques that are simple procedures that work well invivo. Electroporation, using electrical methods to change thepermeability of the cellular membrane, has been successful for some invitro therapies for insertion of genes into cells

There have been some attempts at targeted delivery of DNA for specificcells that relied upon the presence of receptors for glycoproteins. Thedelivery system used polycations, such as polylysine, that werenoncovalently bonded to DNA, and that were also covalently bonded to aligand. Such use of covalently bonding of the polycations to a liganddoes not allow for the disassembly of the delivery system once thecellular internalization mechanisms begin. This large complex,covalently bonded delivery system is very unlike the way nucleic acidsare naturally found within cells.

Simple, efficient delivery systems for delivery of specific therapeuticagents to specific sites in the body for the treatment of diseases orpathologies or for the detection of such sites are not currentlyavailable. For example, current treatments for cancer includeadministration of chemotherapeutic agents and other biologically activefactors such as cytokines and immune factors that impact the entireorganism. The side effects include organ damage, loss of senses such astaste and feel, and hair loss. Such therapies provide treatment for thecondition, but also require many adjunct therapies to treat the sideeffects.

What is needed are compositions and methods for delivery systems ofagents that effect the desired cells or site. These delivery systemscould be used for delivery to specific cells of agents of all types,including detection and therapeutic agents. What is also needed aredelivery systems that do not cause unwanted side effects in the entireorganism.

SUMMARY OF THE INVENTION

The present invention comprises compositions and methods for deliverysystems of agents, including therapeutic compounds, pharmaceuticalagents, drugs, detection agents, nucleic acid sequences and biologicalfactors. In general, these vector compositions comprise a colloidalmetal sol, derivatized PEG (polyethylene glycol) and an agent, andmethods and compositions for making such colloidal metal solcompositions.

The vector compositions are particularly useful in detection ortreatment of solid tumors. Preferred compositions of the presentinvention comprise vectors comprising colloidal metal sols, preferablygold metal sols, associated with derivatized-PEG, preferably thiol-PEG,and also comprise one or more agents that aid in specific targeting ofthe vector or have therapeutic effects or can be detected.

The present invention comprises methods of delivery by administering thecompositions of the invention by known methods such as injection ororally, wherein the compositions are delivered to specific cells ororgans. In one embodiment, the present invention comprises methods fortreating diseases, such as cancer or solid tumors, by administering thecompositions of the present invention comprising agents that are knownfor the treatment of such diseases. Another embodiment comprises vectorcompositions comprising derivatized PEG, TNF (Tumor Necrosis Factor) andanti-cancer agents, associated with colloidal metal particles. Inanother embodiment, the present invention comprises methods for genetherapy by administering the compositions of the present inventioncomprising agents that are used for gene therapy, such asoligonucleotides, antisense, vectors, ribozymes, DNA, RNA, senseoligonucleotides, and nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a mixing apparatus used to prepare vectors.

FIG. 2 is graph showing saturation binding of TNF to colloidal gold.

FIG. 3A is a graph showing the effect of TNF:gold binding ratios on thesafety of the cAu-TNF vector.

FIG. 3B is a graph showing the effects of a TNF:gold binding ratios onthe safety of the cAu-TNF vector.

FIG. 3C is a chart showing the anti-tumor efficacy a cAu-TNF and nativeTNF.

FIG. 3D is a chart showing TNF distribution profiles after 1 hour.

FIG. 3E is a chart showing TNF distribution profiles after 8 hours.

FIG. 3F is a graph of pharmacokinetic profiles of native TNF and acAu-TNF vector in MC38 tumor-burdened C57/BL6 mice.

FIG. 4, A-C, shows liver and spleen of mice treated with PT-cAu-TNFvectors, (A), cAU-TNF vectors, and no treatment (C).

FIG. 5A is a graph showing gold distribution in various organs.

FIG. 5B is a graph showing TNF pharmacokinetic analysis.

FIG. 5C is a graph showing the intra-tumor TNF distribution over time.

FIG. 5D is a chart comparing the intra-tumor TNF concentrations withdifferent vectors.

FIGS. 5E and F are graphs showing the distribution of TNF in variousorgans over time.

FIG. 6A is a graph comparing safety and efficacy of native TNF orPT-cAu-TNF vectors.

FIG. 6B is a graph comparing Native TNF and 20K-PT-cAU-TNF safety andefficacy.

FIG. 6C is a graph comparing Native TNF and 30K-PT-cAU-TNF safety andefficacy.

FIG. 7 is a schematic of a vector having multiple agents.

FIG. 8 is a schematic of an embodiment of a capture method for detectinga vector.

FIG. 9 is a graph showing TNF- and END-captured vectors exhibiting thepresence of the second agent.

FIG. 10 is a graph showing TNF- and END-captured vectors exhibiting thepresence of the second agent.

DETAILED DESCRIPTION

The present invention comprises compositions and methods for thedelivery of agents. The present invention also comprises methods formaking the compositions and administering the compositions in vitro andin vivo. In general, the present invention contemplates compositionscomprising metal sol particles associated with any or all of thefollowing components alone or in combinations: active agents, detectionagents, targeting molecules, integrating molecules, and one or moretypes PEG or derivatized PEGs.

The delivery of agents is used for detection or treatment of specificcells or tissues. For example, the present invention is used for imagingspecific tissue, such as solid tumors. The delivery of agents is usedfor treatments of biological conditions, including, but not limited to,chronic and acute diseases, maintenance and control of the immune systemand other biological systems, infectious diseases, vaccinations,hormonal maintenance and control, cancer, solid tumors and angiogenicstates. Such delivery may be targeted to specific cells or cell types,or the delivery may be less specifically provided to the body, inmethods that allow for low level release of the agent or agents in anontoxic manner. Descriptions and uses of metal sol compositions aretaught in U.S. Pat. No. 6,274,552; and related patent applications, U.S.patent application Ser. Nos. 09/808,809; 09/935,062; 09/189,748;09/189,657,and 09/803,123; and U.S. Provisional Patent Applications60/287,363, all of which are herein incorporated in their entireties.

The present invention is directed to methods and compositions comprisingcolloidal metals as vectors for delivery of agents. Specifically,preferred compositions are used in methods of treatment or detectioncomprising accumulation of one or more types of vectors in a solidtumor. Methods for treating solid tumors comprise administeringcolloidal metal sol compositions comprising PEG, preferablyderivatized-PEG, more preferably, thiol-derivatized polyethyleneglycols. Though not wishing to be bound by any particular theory, it isthought that use of such compositions results in the vector compositiontrafficking to and accumulating in tumors. In the absence of targetingmolecules or active agents, a derivatized PEG colloidal metal vectortraffics to the tumor and is sequestered there. All methods ofadministration are contemplated by the present invention, though themost preferable routes of administration are intravenous or oral. Whenadministered, preferably intravenously or orally, the colloidal vectorsare found in or associated with a tumor.

The compositions of the invention preferably comprise a colloidal metalsol, derivatized compounds and one or more agents. The agents may bebiologically active agents that can be used in therapeutic applicationsor the agents may be agents that are useful in detection methods. Inpreferred embodiments, one or more agents are admixed, associated withor bound directly or indirectly to the colloidal metal. Admixing,associating and binding includes covalent and ionic bonds and otherweaker or stronger associations that allow for long term or short termassociation of the derivatized-PEG, agents, and other components witheach other and with the metal sol particles.

In yet another embodiment, the compositions also comprise one or moretargeting molecules admixed, associated with or bound to the colloidalmetal. The targeting molecule can be bound directly or indirectly to themetal particle. Indirect binding includes binding through molecules suchas polylysines or other integrating molecules or any association with amolecule that binds to both the targeting molecule and either the metalsol or another molecule bound to the metal sol.

Any colloidal metal can be used in the present invention. Colloidalmetal includes any water-insoluble metal particle or metallic compounddispersed in liquid water, a hydrosol or a metal sol. The colloidalmetal may be selected from the metals in groups IA, IB, IIB and IIIB ofthe periodic table, as well as the transition metals, especially thoseof group VIII. Preferred metals include gold, silver, aluminum,ruthenium, zinc, iron, nickel and calcium. Other suitable metals alsoinclude the following in all of their various oxidation states: lithium,sodium, magnesium, potassium, scandium, titanium, vanadium, chromium,manganese, cobalt, copper, gallium, strontium, niobium, molybdenum,palladium, indium, tin, tungsten, rhenium, platinum, and gadolinium. Themetals are preferably provided in ionic form, derived from anappropriate metal compound, for example the Al³⁺, Ru³⁺, Zn²⁺, Fe³⁺, Ni²⁺and Ca²⁺ ions.

A preferred metal is gold, particularly in the form of Al³⁺. Anespecially preferred form of colloidal gold is HAuCl₄. In oneembodiment, the colloidal gold particles have a negative charge at anapproximately neutral pH. It is thought that this negative chargeprevents the attraction and attachment of other negatively chargedmolecules. In contrast, positively charged molecules are attracted toand bind to the colloidal gold particle. The colloidal gold is employedin the form of a sol which contains gold particles having a range ofparticle sizes, though a preferred size is a particle size ofapproximately 30 to 40 nm.

Another preferred metal is silver, particularly in a sodium boratebuffer, having the concentration of between approximately 0.1% and0.001%, and most preferably, approximately a 0.01% solution. Preferably,the color of such a colloidal silver solution is yellow and thecolloidal particles range from 1 to 40 nm. Such metal ions may bepresent in the complex alone or with other inorganic ions.

The agent of the present invention can be any compound, chemical,therapeutic agent, pharmaceutical agent, drug, biological factors,fragments of biological molecules such as antibodies, proteins, lipids,nucleic acids or carbohydrates; nucleic acids, antibodies, proteins,lipids, nutrients, cofactors, nutriceuticals, anesthetic, detectionagents or an agent that has an effect in the body. Such detection andtherapeutic agents and their activities are known to those of ordinaryskill in the art.

The following are non-limiting examples of some of the agents that canbe used in the present invention. One type of agent that can be employedin the present invention includes biological factors including, but notlimited to, cytokines, growth factors, fragments of larger moleculesthat have activity, neurochemicals, and cellular communicationmolecules. Examples of such agents include, but are not limited to,Interleukin-1 (“IL-1”), Interleukin-2 (“IL-2”), Interleukin-3 (“IL-3”),Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Interleukin-12 (“IL-12”),Interleukin-13 (“IL-13”), Interleukin-15 (“IL-15”), Interleukin-16(“IL-16”), Interleukin-17 (“IL-17”), Interleukin-18 (“IL-18”), Type IInterferon, Type II Interferon, Tumor Necrosis Factor (“TNFα”),Transforming Growth Factor-α (“TGF-α”), Lymphotoxin, MigrationInhibition Factor, Granulocyte-Macrophage Colony-Stimulating Factor(“CSF”), Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelialgrowth factor (“VEGF”), Angiogenin, transforming growth factor-β(“TGF-β”), fibroblast growth factor, angiostatin, endostatin, GABA, andacetyl choline.

Another type of agent includes hormones. Examples of such hormonesinclude, but are not limited to, growth hormone, insulin, glucagon,parathyroid hormone, luteinizing hormone, follicle stimulating hormone,luteinizing hormone releasing hormone, estrogen, testosterone,dihydrotestoerone, estradiol, prosterol, progesterone, progestin,estrone, other sex hormones, and derivatives and analogs of hormones.

Yet another type of agent includes pharmaceuticals. Any type ofpharmaceutical agent can be employed in the present invention. Forexample, antiinflammatory agents such as steroids and nonsteroidalantiinflammatory agents, soluble receptors, antibodies, antibiotics,analgesics, angiogenic and anti-angiogenic agents, and COX-2 inhibitors,can be employed in the present invention. Chemotherapeutic agents are ofparticular interest in the present invention. Nonlimiting examples ofsuch agents include taxol, paclitaxel, taxanes, vinblastin, vincristine,doxorubicin, acyclovir, cis-platin and tacrine.

Immunotherapy agents are also of particular interest in the presentinvention. Nonlimiting examples of immunotherapy agents, includeinflammatory agents, biological factors, immune regulatory proteins, andimmunotherapy drugs, such as AZT and other derivatized or modifiednucleotides. Small molecules can also be employed as agents in thepresent invention.

Another type of agent includes nucleic acid-based materials. Examples ofsuch materials include, but are not limited to, nucleic acids,nucleotides, DNA, RNA, tRNA, mRNA, sense nucleic acids, antisensenucleic acids, ribozymes, DNAzymes, protein/nucleic acid compositions,SNPs, oligonucleotides, vectors, viruses, plasmids, transposons, andother nucleic acid constructs known to those skilled in the art.

Other agents that can be employed in the invention include, but are notlimited to, lipid A, phospholipase A2, endotoxins, staphylococcalenterotoxin B and other toxins, heat shock proteins, carbohydratemoieties of blood groups, Rh factors, cell surface receptors,antibodies, cancer cell specific antigens; such as MART, MAGE, BAGE, andHSPs (Heat Shock Proteins), radioactive metals or molecules, detectionagents, enzymes and enzyme co-factors.

Of particular interest are detection agents such as dyes or radioactivematerials that can be used for visualizing or detecting the sequesteredcolloidal metal vectors. Fluorescent, chemiluminescent, heat sensitive,opaque, beads, magnetic and vibrational materials are also contemplatedfor use as detectable agents that are associated or bound to colloidalmetals in the compositions of the present invention.

Other examples of agents and organisms that are affected by treatmentmethods of the present invention are found in the following table. Thistable is not limiting in that other agents, such as the pharmaceuticalequivalents of the following agents, are contemplated by the presentinvention.

TABLE I Organisms and Selected Active Agents BACTERIA Mycobacteriumtuberculosis Isoniazid, rifampin, ethambutol, pyrazinamide,streptomycin, clofazimine, rifabutin, fluoroquinolones such as ofloxacinand sparfloxacin Mycobacterium avium Rifabutin, rifampin, azithromycin,clarithromycin, fluoroquinolones Mycobacterium leprae Dapsone Chlamydiatrachomatis Tetracycline, doxycyline, erythromycin, ciprofloxacinChlamydia pneumoniae Doxycycline, erythromycin Listeria monocytogenesAmpicillin FUNGI Candida albicans Amphotericin B, ketoconazole,fluconazole Cryptococcus neoformans Amphotericin B, ketoconazole,fluconazole PROTOZOA Toxoplasma gondii Pyrimethamine, sulfadiazine,clindamycin, azithromycin, clarithromycin, atovaquone Pneumocystiscarinii Pentamidine, atovaquone Cryptosporidium sp. Paromomycin,diclazaril VIRUS Herpes simplex virus type 1 Acyclovir, trifluorouridineand other and type 2 antiviral nucleoside analogs, foscornat, antisenseoligonucleotides, and triplex-specific DNA sequences CytomegalovirusFoscarnet, ganciclovir HIV AZT, DDI, DDC, foscarnat, viral proteaseinhibitors, peptides, antisense oligonucleotides, triplex and othernucleic acid sequences Influenza virus types A & B Ribavirin Respiratorysyncytial virus Ribavirin Varizella zoster virus Acyclovir

Targeting molecules are also components of compositions of the presentinvention. One or more targeting molecules may be directly or indirectlyattached, bound or associated with the colloidal metal. These targetingmolecules can be directed to specific cells or cell types, cells derivedfrom a specific embryonic tissue, organs or tissues. Such targetingmolecules include any molecules that are capable of selectively bindingto specific cells or cell types. In general, such targeting moleculesare one member of a binding pair and as such, selectively bind to theother member. Such selectivity may be achieved by binding to structuresfound naturally on cells, such as receptors found in cellular membranes,nuclear membranes or associated with DNA. The binding pair member mayalso be introduced synthetically on the cell, cell type, tissue ororgan. Targeting molecules also include receptors or parts of receptorsthat may bind to molecules found in the cellular membranes or free ofcellular membranes, ligands, antibodies, antibody fragments, enzymes,cofactors, substrates, and other binding pair members known to thoseskilled in the art. Targeting molecules may also be capable of bindingto multiple types of binding partners. For example, the targetingmolecule may bind to a class or family of receptors or other bindingpartners. The targeting molecule may also be an enzyme substrate orcofactor capable of binding several enzymes or types of enzymes.

Specific examples of targeting molecules include, but are not limitedto, Interleukin-1 (“IL-1”), Interleukin-2 (“IL-2”), Interleukin-3(“IL-3”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6(“IL-6”), Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Interleukin-12 (“IL-12”),Interleukin-13 (“IL-13”), Interleukin-15 (“IL-15”), Interleukin-16(“IL-16”), Interleukin-17 (“IL-17”), Interleukin-18 (“IL-18”), lipid A,phospholipase A2, endotoxins, staphylococcal enterotoxin B and othertoxins, Type I Interferon, Type II Interferon, Tumor Necrosis Factor(“TNFα”), Transforming Growth Factor-α “TGF-α”), Lymphotoxin, MigrationInhibition Factor, Granulocyte-Macrophage Colony-Stimulating Factor(“CSF”), Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelialgrowth factor (“VEGF”), Angiogenin, transforming growth factor-β(“TGF-β”), carbohydrate moieties of blood groups, Rh factors, fibroblastgrowth factor and other inflammatory and immune regulatory proteins,hormones, such as growth hormone, insulin, glucagon, parathyroidhormone, luteinizing hormone, follicle stimulating hormone, andluteinizing hormone releasing hormone, cell surface receptors,antibodies, nucleic acids, nucleotides, DNA, RNA, sense nucleic acids,antisense nucleic acids, cancer cell specific antigens, MART, MAGE,BAGE, and HSPs (Heat Shock Proteins), mutant p53; tyrosinase; antoimmuneantigens; receptor proteins, glucose, glycogen, phospholipids, andmonoclonal and/or polyclonal antibodies, basic fibroblast growth factor,enzymes, cofactors and enzyme substrates.

The integrating molecules used in the present invention can either bespecific binding integrating molecules, such as members of a bindingpair, or can be nonspecific binding integrating molecules that bind lessspecifically. An integrating molecule is defined by its function ofproviding a site for binding or associating two entities. One entity cancomprise a metal sol particle, and the other entity can comprise one ormore active agents or one or more targeting molecules, or both orcombinations thereof. The compositions of the present invention cancomprise one or more integrating molecules.

An example of a nonspecific binding-integrating molecule is polycationicmolecules such as polylysine or histones that are useful in bindingnucleic acids. Polycationic molecules are known to those skilled in theart and include, but are not limited to, polylysine, protamine sulfate,histones or asialoglycoproteins. The present invention also contemplatesthe use of synthetic molecules that provide for binding one or moreentities to the metal particles.

Specific binding-integrating molecules comprise any members of bindingpairs that can be used in the present invention. Such binding pairs areknown to those skilled in the art and include, but are not limited to,antibody-antigen pairs, enzyme-substrate pairs, receptor-ligand pairs,and streptavidin-biotin. In addition to such known binding pairs, novelbinding pairs may be specifically designed. A characteristic of bindingpairs is the binding between the two members of the binding pair.Another desired characteristic of the binding partners is that onemember of the pair is capable of binding or being bound to one or moreof an agent or a targeting molecule, and the other member of the pair iscapable of binding to the metal particle.

Another component of the compositions of the present invention comprisesglycol compounds, preferably polyethylene glycol (PEG), more preferablyderivatized PEG. The present invention comprises compositions comprisingderivatized PEG, wherein the PEG is 5,000 to 30,000 MW. Derivatized PEGcompounds are commercially available from sources such as ShearwaterCorporation, Huntsville, Ala. PEG compounds may be difunctional ormonofunctional, such as methoxy-PEG (mPEG). Activated derivatives oflinear and branched PEGs are available in a variety of molecularweights. As used herein, the term “derivatized PEG(s)” or “PEGderivative(s)” means any polyethylene glycol molecule that has beenaltered with either addition of functional groups, chemical entities, oraddition of other PEG groups to provide branches from a linear molecule.Such derivatized PEGs can be used for conjugation with biologicallyactive compounds, preparation of polymer grafts, or other functionsprovided by the derivatizing molecule.

One type of PEG derivative is a polyethylene glycol molecule withprimary amino groups at one or both of the termini. A preferred moleculeis methoxy PEG with an amino group on one terminus. Another type of PEGderivative includes electrophilically activated PEG. These PEGs are usedfor attachment of PEG or methoxy PEG (mPEG), to proteins, liposomes,soluble and insoluble polymers and a variety of molecules.Electrophilically active PEG derivatives include succinimide of PEGpropionic acid, succinimide of PEG butanoate acid, multiple PEGsattached to hydroxysuccinimide or aldehydes, mMPEG double esters(mPEG-CM-HBA-NHS), MPEG benzotriazole carbonate, and MPEGpropionaldehyde, niPEG acetaldehyde diethyl acetal.

A preferred type of derivatized PEG comprises thiol derivatized PEGs, orsulfhydryl-selective PEGs. Branched, forked or linear PEGs can be usedas the PEG backbone that has a molecular weight range of 5,000 to 40,000mw. Preferred thiol derivatized PEGs comprise PEG with maleimidefunctional group to which a thiol group can be conjugated. A preferredthiol-PEG is methoxy-PEG-maleimide, with PEG mw of 5,000 to 40,000.

Use of heterofunctional PEGs, as a derivatized PEG, is also contemplatedby the present invention. Heterofunctional derivatives of PEG have thegeneral structure X-PEG-Y. When the X and Y are functional groups thatprovide conjugation capabilities, many different entities can be boundon either or both termini of the PEG molecule. For example, vinylsulfoneor maleimide can be X and an NHS ester can be Y. For detection methods,X and/or Y can be fluorescent molecules, radioactive molecules,luminescent molecules or other detectable labels. Heterofunctional PEGor monofunctional PEGs can be used to conjugate one member of a bindingpair, such as PEG-biotin, PEG-Antibody, PEG-antigen, PEG-receptor,PEG-enzyme or PEG-enzyme substrate. PEG can also be conjugated to lipidssuch as PEG-phospholipids.

One or more agents of the compositions of the present invention can bebound directly to the colloidal metal particles or can be boundindirectly to the colloidal metal through one or more integratingmolecules. One method of preparing colloidal metal sols of the presentinvention uses the method described by Horisberger, (1979), which isincorporated by reference herein. In embodiments where an integratingmolecule is employed, the integrating molecule is bound to, admixed orassociated with the metal sol. The agent may be bound to, admixed orassociated with the integrating molecule prior to the binding, admixingor associating of the integrating molecule with the metal, or may bebound, admixed or associated after the binding of the integratingmolecule to the metal.

General methods for binding agents to metal sols comprise the followingsteps. A solution of the agent is formed in a buffer or solvent, such asdeionized water (diH₂O). The appropriate buffer or solvent will dependupon the agent to be bound. Determination of the appropriate buffer orsolvent for a given agent is within the level of skill of the ordinaryartisan. Determining the pH necessary to bind an optimum amount of agentto metal sol is known to those skilled in the art. The amount of agentbound can be determined by quantitative methods for determiningproteins, therapeutic agents or detection agents, such as ELISA orspectrophotometric methods. Where integrating molecules are employed inthe present invention, the binding pH and saturation level of theintegrating molecule is also considered in preparing the compositions.For example, where the integrating molecule is a member of a bindingpair, such as Streptavidin-biotin, the binding pH for streptavidin orbiotin is determined and the concentration of the streptavidin or biotinbound can also determined.

When the vector composition comprises an integrating molecule, the agentmay be bound to a member of the binding pair which is functioning as anintegrating molecule, such as biotin, by conventional methods known inthe art. The biotinylated agent can then be added to the colloidal goldcomposition comprising the integrating molecule, streptavidin. Thebiotin binds specifically to the streptavidin providing an indirect bondbetween the colloidal gold and the active agent.

One method of binding an agent to metal sols comprises the followingsteps, though for clarity purposes only, the methods disclosed refer tobinding an agent, TNF, to a metal sol, colloidal gold. An apparatus wasused that allows interaction between the particles in the colloidal goldsol and TNF in a protein solution. A schematic representation of theapparatus is shown in FIG. 1. This apparatus maximizes the interactionof unbound colloidal gold particles with the protein to be bound, TNF,by reducing the mixing chamber to a small volume. This apparatus enablesthe interaction of large volumes of gold sols with large volumes of TNFto occur in the small volume of a T connector. In contrast, adding asmall volume of protein to a large volume of colloidal gold particles isnot a preferred method to ensure uniform protein binding to the goldparticles. Nor is the opposite method of adding small volumes ofcolloidal gold to a large volume of protein. Physically, the colloidalgold particles and the protein, TNF are forced into a T-connector by asingle peristaltic pump that draws the colloidal gold particles and theTNF protein from two large reservoirs. To further ensure proper mixing,an in-line mixer is placed immediately down stream of the T-connector.The mixer vigorously mixes the colloidal gold particles with TNF, bothof which are flowing through the connector at a preferable flow rate ofapproximately 1 L/min.

Prior to mixing with the agent, the pH of the gold sol is adjusted to pH8-9 using 1 N NaOH. Highly purified, lyophilized recombinant human TNFis reconstituted and diluted in 3 mM Tris. Before adding either the solor TNF to their respective reservoirs, the tubing connecting thecontainers to the T-connector is clamped shut. Equal volumes ofcolloidal gold sol and the TNF solution are added to the appropriatereservoirs. Preferred concentrations of agent in the solution range fromapproximately 0.01 to 15 μg/ml, and can be altered depending on theratio of the agent to metal sol particles. Preferred concentrations ofTNF in the solution range from 0.5 to 4 μg/ml and the most preferredconcentration of TNF for the TNF-colloidal gold composition is 0.5μg/ml.

Once the solutions are properly loaded into their respective reservoirs,the peristaltic pump is turned on, drawing the agent solution and thecolloidal gold solution into the T-connector, through the in-line mixer,through the peristaltic pump and into a collection flask. The mixedsolution is stirred in the collection flask for an additional hour ofincubation.

In compositions comprising PEG, whether derivatized or not, the methodsfor making such compositions comprise the following steps, though forclarity purposes only, the methods disclosed refer to adding PEG thiolto a metal sol composition. Any PEG, derivatized PEG composition or anysized PEG compositions or compositions comprising several differentPEGs, can be made using the following steps. Following the 1-hourincubation taught above, a thiol derivatized polyethylene glycol (PEG)solution is added to the colloidal gold/TNF sol. The present inventioncontemplates use of any sized PEG with any derivative group, thoughpreferred derivatized PEGs include mPEG-OPSS/5,000,thiol-PEG-thiol/3,400, mPEG-thiol 5000, and mPEG thiol 20,000(Shearwater Polymers, Inc.). A preferred PEG is mPEG-thiol 5000 at aconcentration of 150 μg/ml in water, pH 5-8. Thus, a 10% v/v of the PEGsolution is added to the colloidal gold-TNF solution. The gold/TNF/PEGsolution is incubated for an additional hour.

The colloidal gold/TNF/PEG solution is subsequently ultrafilteredthrough a 50K MWCO diafiltration cartridge. The 50K retentate andpermeate are measured for TNF concentration by ELISA to determine theamount of TNF bound to the gold particles.

The compositions of the present invention can be administered to invitro and in vivo systems. In vivo administration may include directapplication to the target cells or such routes of administration,including but not limited to formulations suitable for oral, rectal,transdermal, ophthalmic, (including intravitreal or intracameral) nasal,topical (including buccal and sublingual), vaginal or parenteral(including subcutaneous, intramuscular, intravenous, intradermal,intratracheal, and epidural) administration. A preferred methodcomprising administering, via oral or injection routes, an effectiveamount of a composition comprising vectors of the present invention.

The formulations may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques.Pharmaceutical formulation compositions are made by bringing intoassociation the metal sol vectors and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the compositions with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Preferred methods of use of the compositions of the present inventioncomprise targeting the vectors to tumors. Preferred vector compositionscomprise metal sol particles, agents and PEG or derivatized PEGcompositions for delivery to a tumor for therapeutic effects on thetumor or organism or detection of tumors. Such vector compositions mayfurther comprise targeting and/or integrating molecules. Still otherpreferred vector compositions comprise metal sol particles, radioactiveor cytotoxic agents and PEG or derivatized PEG compositions for deliveryof radiation therapies to tumors. Historically, radioactive colloidalgold was used as a cancer therapy, principally for the treatment ofliver cancer due to the anticipated uptake of colloidal gold by theliver cells. Compositions comprising derivatized PEG, preferably PEGthiol, in combination with radioactive colloidal metal particles areused to treat or identify tumors. Alternatively, a vector compositioncomprising a radioactive moiety coupled to a protein that is bound tocolloidal metal, and further comprising derivatized PEG, preferablyPEG-thiol, forming a radioactive vector, is used to treat tumors. Theradioactive vector composition of the present invention is injectedintravenously and traffics to the tumor and is not significantly takenup by the liver. In both compositions, it is believed that the abilityof the PEG thiol to concentrate the radioactive therapy in the tumorincreases treatment efficacy while reducing treatment side effects.

Other preferred vector compositions comprise metal sol particles andPEG, preferably PEG derivatives, for use in methods comprisingadministering the compositions for in vivo imaging and detection oftumors. The compositions may further comprise agents that aid in thedetection and imaging methods. For example, the agents include, but arenot limited to, radioactive, radiation sensitive or reactive, such aslight or heat reactive compounds, chemiluminescent or luminescent agentsor other agents used for detection purposes. Methods of detectioninclude, but are not limited to NMR, CAT or PET scans, visualexamination, colorimetric, radiation detection methods,spectrophotometric, and protein, nucleic acid, polysaccharide or otherbiological agent detection methods.

The present invention comprises compositions for use in methods fordelivery of exogenous nucleic acids or genetic material into cells. Theexogenous genetic material may be targeted to specific cells usingtargeting molecules that are capable of recognizing the specific cellsor specifically targeted to tumors using compositions comprising PEG orderivatized PEG. For example, the targeting molecule is a bindingpartner for a specific receptor on the cells, and after binding, theentire composition may be internalized within the cells. The binding ofthe vector composition may activate cellular mechanisms that alter thestate of the cell, such as activation of secondary messenger moleculeswithin the cell. Thus, in a mixture of different cell types, theexogenous nucleic acids are delivered to cells having the selectedreceptor and cells lacking the receptor are unaffected.

The present invention comprises compositions and methods for thetransfection of specific cells, in vitro or in vivo, for insertion orapplication of agents. One embodiment of such a composition comprisesnucleic acid bound to polycations (nonspecific binding-integratingmolecules) that are bound to colloidal metals. A preferred embodiment ofthe present invention comprises colloidal gold as a platform that iscapable of binding targeting molecules and nucleic acid agents to createa targeted gene delivery vector that employs receptor-mediatedendocytosis of cells to achieve transfection. In a more preferredembodiment, the targeting molecule is a cytokine and the agent isgenetic material such as DNA or RNA. This embodiment may also compriseintegrating molecules such as polycations to which the genetic materialis bound or associated.

In the present invention, the methods comprise the preparation of genedelivery vectors and delivery of the targeted gene delivery vector tothe cells for transfection or therapeutic effects. It is contemplated inthe present invention that the nucleic acids of the compositions may beinternalized and used as detection agents or for genetic therapeuticeffects, or the nucleic acids can be translated and expressed by thecell. The expression products can be any known to those skilled in theart and includes but is not limited to functioning proteins, productionof cellular products, enzymatic activity, export of cellular products,production of cellular membrane components, or nuclear components. Themethods of delivery to the targeted cells may be such methods as thoseused for in vitro techniques such as with cellular cultures, or thoseused for in vivo administration. In vivo administration may includedirect application to the cells or such routes of administration as usedfor humans, animals or other organism, preferably intravenous or oraladministration. The present invention also contemplates cells that havebeen altered by the compositions of the present invention and theadministration of such cells to other cells, tissues or organisms, in invitro or in vivo methods.

The present invention comprises compositions and methods for enhancingan immune response and increasing vaccine efficacy through thesimultaneous or sequential targeting of specific immune cells usingcompositions directed to specific immune components. The compositionscan also be used in methods for imaging or detecting immune cells. Thesemethods comprise vector compositions that are capable of effecting theimmune system, and include colloidal metals associated with at least oneof the following components, targeting molecules, agents, integratingmolecules, one or more types of PEG or derivatized PEGs. Thecompositions may also comprise specific immune components, such as cellsincluding, but not limited to, antigen presenting cells (APCs), such asmacrophages and dendritic cells, and lymphocytes, such as B cells and Tcells, which have been or are individually effected by one or morecomponent-specific immunostimulating agents.

Examples of component-specific immunostimulating molecules include, butare not limited to, Interleukin-1 (“IL-1”), Interleukin-2 (“IL-2”),Interleukin-3 (“IL-3”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”),Interleukin-6 (“IL-6”), Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”),Interleukin-10 (“IL-10”), Interleukin-11 (“IL-11”), Interleukin-12(“IL-12”), Interleukin-13 (“IL-13”), lipid A, phospholipase A2,endotoxins, staphylococcal enterotoxin B and other toxins, Type IInterferon, Type II Interferon, Tumor Necrosis Factor (“TNF-α”),Transforming Growth Factor-β(“TGF-β” Lymphotoxin, Migration InhibitionFactor, Granulocyte-Macrophage Colony-Stimulating Factor (“CSF”),Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelial growthfactor (“VEGF”), Angiogenin, transforming growth factor (“TGF-α”), heatshock proteins, carbohydrate moieties of blood groups, Rh factors,fibroblast growth factor, and other inflammatory and immune regulatoryproteins, nucleotides, DNA, RNA, mRNA, sense, antisense, cancer cellspecific antigens; such as MART, MAGE, BAGE; flt3 ligand/receptorsystem; B7 family of molecules and receptors; CD 40 ligand/receptor; andimmunotherapy drugs, such as AZT; and angiogenic and anti-angiogenicdrugs, such as angiostatin, endostatin, and basic fibroblast growthfactor, or vascular endothelial growth factor (VEGF).

An especially preferred embodiment provides methods for activation ofthe immune response using vector compositions comprising agentscomprising a specific antigen in combination with a component-specificimmunostimulating agent. As used herein, component-specificimmunostimulating agent means an agent that is specific for a componentof the immune system, such as a B or T cell, and that is capable ofaffecting that component, so that the component has an activity in theimmune response. The component-specific immunostimulating agent may becapable of affecting several different components of the immune system,and this capability may be employed in the methods and compositions ofthe present invention. The agent may be naturally occurring or can begenerated or modified through molecular biological techniques or proteinreceptor manipulations.

The activation of the component in the immune response may result in astimulation or suppression of other components of the immune response,leading to an overall stimulation or suppression of the immune response.For ease of expression, stimulation of immune components is describedherein, but it is understood that all responses of immune components arecontemplated by the term stimulation, including but not limited tostimulation, suppression, rejection and feedback activities.

The immune component that is effected may have multiple activities,leading to both suppression and stimulation or initiation or suppressionof feedback mechanisms. The present invention is not to be limited bythe examples of immunological responses detailed herein, butcontemplates component-specific effects in all aspects of the immunesystem.

The activation of each of the components of the immune system may besimultaneous, sequential, or any combination thereof. In one embodimentof a method of the present invention, multiple component-specificimmunostimulating agents are administered simultaneously. In thismethod, the immune system is simultaneously stimulated with multipleseparate preparations, each containing a vector composition comprising acomponent-specific immunostimulating agent. Preferably, the vectorcomposition comprises the component-specific immunostimulating agentassociated with colloidal metal. More preferably, the compositioncomprises the component-specific immunostimulating agent associated withcolloidal metal of one sized particle or of different sized particlesand an antigen. Most preferably, the composition comprises thecomponent-specific immunostimulating agent associated with colloidalmetal of one sized particle or of differently sized particles, antigenand PEG or PEG derivatives.

Component-specific immunostimulating agents provides a specificstimulatory, up regulation, effect on individual immune components. Forexample, Interleukin-1β (IL-1β) specifically stimulates macrophages,while TNF-α (Tumor Necrosis Factor alpha) and Flt-3 ligand specificallystimulate dendritic cells. Heat killed Mycobacterium butyricum andInterleukin-6 (IL-6) are specific stimulators of B cells, andInterleukin-2 (IL-2) is a specific stimulator of T cells. Vectorcompositions comprising such component-specific immunostimulating agentsprovide for specific activation of macrophages, dendritic cells, B cellsand T cells, respectively. For example, macrophages are activated when avector composition comprising the component-specific immunostimulatingagent IL-1β is administered. A preferred composition is IL-1β inassociation with colloidal metal, and a most preferred composition isIL-1β in association with colloidal metal and an antigen to provide aspecific macrophage response to that antigen. Vector compositions canfurther comprise targeting molecules, integrating molecules, PEGs orderivatized PEGs.

Many elements of the immune response may be necessary for an effectiveimmune response to an antigen. An embodiment of a method of simultaneousstimulation is to administer four separate preparations of compositionsof component-specific immunostimulating agents comprising 1) IL-1β formacrophages, 2) TNF-α and Flt-3 ligand for dendritic cells, 3) IL-6 forB cells, and 4) IL-2 for T cells. Each component-specificimmunostimulating agent vector composition may be administered by anyroutes known to those skilled in the art, and all may use the same routeor different routes, depending on the immune response desired.

In another embodiment of the methods and compositions of the presentinvention, the individual immune components are activated sequentially.For example, this sequential activation can be divided into two phases,a primer phase and an immunization phase. The primer phase comprisesstimulating APCs, preferably macrophages and dendritic cells, while theimmunization phase comprises stimulating lymphocytes, preferably B cellsand T cells. Within each of the two phases, activation of the individualimmune components may be simultaneous or sequential. For sequentialactivation, a preferred method of activation is administration of vectorcompositions that cause activation of macrophages followed by dendriticcells, followed by B cells, followed by T cells. A most preferred methodis a combined sequential activation comprising the administration ofvector compositions which cause simultaneous activation of themacrophages and dendritic cells, followed by the simultaneous activationof B cells and T cells. This is an example of methods and compositionsof multiple component-specific immunostimulating agents to initiateseveral pathways of the immune system.

The methods and compositions of the present invention can be used toenhance the effectiveness of any type of vaccine. The present methodsenhance vaccine effectiveness by targeting specific immune componentsfor activation. Vector compositions comprising at leastcomponent-specific immunostimulating agents in association withcolloidal metal and antigen are used for increasing the contact betweenantigen and the specific immune component, such as macrophages, B or Tcells. Examples of diseases for which vaccines are currently availableinclude, but are not limited to, cholera, diphtheria, Haemophilus,hepatitis A, hepatitis B, influenza, measles, meningitis, mumps,pertussis, small pox, pneumococcal pneumonia, polio, rabies, rubella,tetanus, tuberculosis, typhoid, Varicella-zoster, whooping cough, andyellow fever.

The combination of routes of administration and the vector compositionsfor delivering the antigen to the immune system is used to create thedesired immune response. The present invention also comprises methodsand compositions comprising various compositions of packaging systems,such as liposomes, microcapsules, or microspheres, that can providelong-term release of immune stimulating vector compositions. Thesepackaging systems act as internal depots for holding antigen and slowlyreleasing antigen for immune system activation. For example, a liposomemay be filled with a vector composition comprising the agents of anantigen and component-specific immunostimulating agent, bound to orassociated with a colloidal metal. Additional combinations are colloidalgold particles studded with agents such as viral particles which are theactive vaccine candidate or are packaged to contain DNA for a putativevaccine. The vector may also comprise one or more targeting molecules,such as a cytokine, integrating molecules and PEG derivatives, and thevector is then used to target the virus to specific cells. Furthermore,one could use a fusion protein vaccine, which targets two or morepotential vaccine candidates, and provide a vector composition vaccinethat provides protection against two or more infectious microorganisms.The compositions may also include immunogens, which have been chemicallymodified by the addition of polyethylene glycol which may release thematerial slowly.

The compositions comprising a metal particle and the agents comprisingone or more antigens and one or more component-specificimmunostimulating agents, and one or more of integrating and targetingmolecules and PEG or derivatives of PEG, may be packaged in a liposomeor a biodegradable polymer. The vector composition is slowly releasedfrom the liposome or biodegradable polymer and is recognized by theimmune system as foreign and the specific component to which thecomponent-specific immunostimulating agent is directed activates orsuppresses the immune system. For example, the cascade of the immuneresponse is activated more quickly by the presence of thecomponent-specific immunostimulating agent and the immune response isgenerated more quickly and more specifically.

Other methods and compositions contemplated in the present inventioninclude using compositions of metal particles and agents comprisingantigens and component-specific immunostimulating agents, which may alsocomprise integrating and targeting molecules, in which the colloidalmetal particles have different sizes. The compositions may furthercomprise PEG or derivatives of PEG. Sequential administration ofcomponent-specific immunostimulating agents may be accomplished in a onedose administration by use of differently sized colloidal metalparticles. One dose would include multiple independentcomponent-specific immunostimulating agents an antigen and thecombination could be associated with a differently sized or the samesized colloidal metal particle. Thus, simultaneous administration wouldprovide sequential activation of the immune components to yield a moreeffective vaccine and more protection for the population. Other types ofsuch single-dose administration with sequential activation could beprovided by combinations of differently sized or same sized colloidalmetal vector compositions and liposomes or biodegradable polymers, orliposomes or biodegradable polymers filled with differently sized orsame-sized colloidal metal vector compositions.

Use of such vaccination systems as described above are important inproviding vaccines that can be administered in one dose. One doseadministration is important in treating animal populations such aslivestock or wild populations of animals. One dose administration isvital in treatment of populations for whom healthcare is rarelyaccessible such as the poor, homeless, rural residents or persons indeveloping countries that have inadequate health care. Many persons, inall countries, do not have access to preventive types of health care,such as vaccination. The reemergence of infectious diseases, such astuberculosis, has increased the demand for vaccines that can be givenonce and still provide long-lasting, effective protection. Thecompositions and methods of the present invention provide such effectiveprotection.

The methods and compositions of the present invention can also be usedto treat diseases in which an immune response occurs, by stimulating orsuppressing components that are a part of the immune response. Examplesof such diseases include, but are not limited to, Addison's disease,allergies, anaphylaxis, Bruton's syndrome, cancer, including solid andblood borne tumors, eczema, Hashimoto's thyroiditis, polymyositis,dermatomyositis, type 1 diabetes mellitus, acquired immune deficiencysyndrome, transplant rejection, such as kidney, heart, pancreas, lung,bone, and liver transplants, Graves' disease, polyendocrine autoimmunedisease, hepatitis, microscopic polyarteritis, polyarteritis nodosa,pemphigus, primary biliary cirrhosis, pernicious anemia, coeliacdisease, antibody-mediated nephritis, glomerulonephritis, rheumaticdiseases, systemic lupus erthematosus, rheumatoid arthritis,seronegative spondylarthritides, rhinitis, sjogren's syndrome, systemicsclerosis, sclerosing cholangitis, Wegener's granulomatosis, dermatitisherpetiformis, psoriasis, vitiligo, multiple sclerosis,encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis,Lambert-Eaton syndrome, sclera, episclera, uveitis, chronicmucocutaneous candidiasis, urticaria, transient hypogammaglobulinemia ofinfancy, myeloma, X-linked hyper IgM syndrome, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune hemolytic anemia, autoimmunethrombocytopenia, autoimmune neutropenia, Waldenstrom'smacroglobulinemia, amyloidosis, chronic lymphocytic leukemia, andnon-Hodgkin's lymphoma.

The vector compositions of the present invention comprise agentscomprising component-specific immunostimulating agents. A compositionmay comprise one component-specific immunostimulating agent or multiplecomponent-specific immunostimulating agents. Preferred embodiments ofthe vector compositions comprise agents comprising component-specificimmunostimulating agents in association with colloidal metals. Morepreferred embodiments comprise compositions comprising agents comprisingone or more antigens and component-specific immunostimulating agents inassociation with colloidal metals and at least one of the following, PEGor derivatives of PEG, integrating molecules and targeting molecules forspecifically targeting the effect of the component-specificimmunostimulating agents, including, but not limited to, antigens,receptor molecules, nucleic acids, pharmaceuticals, chemotherapy agents,and carriers. The compositions of the present invention may be deliveredto the immune components in any manner. In one embodiment, the agents,comprising an antigen and a component-specific immunostimulating agent,are bound to a colloidal metal in such a manner that a colloidal metalparticle is associated with both the antigen and the immunostimulatingagent.

The present invention includes presentation of agents such as antigenand component-specific immunostimulating agents in a variety ofdifferent delivery platforms or carrier combinations. For example, apreferred embodiment includes administration of a vector compositioncomprising a metal colloid particle bound to agents such as an antigenand component-specific immunostimulating agents in a liposome orbiodegradable polymer carrier. Additional combinations are colloidalgold particles associated with agents such as viral particles which arethe vaccine antigen or which are viable viral particles containingnucleic acids that produce antigens for a vaccine. The vectorcompositions may also comprise targeting molecules such as a cytokine ora selected binding pair member which is used to target the virus tospecific cells, and further comprises other elements taught herein suchas integrating molecules or PEG or PEG derivatives Such embodimentsprovide for a vaccine preparation that slowly releases antigen to theimmune system for a prolonged response. This type of vaccine isespecially beneficial for one-time administration of vaccines. All typesof carriers, including but not limited to liposomes and microcapsulesare contemplated in the present invention.

Toxicity Reduction and Vaccine Administration

The present invention comprises compositions and methods foradministering factors that, when the factors are present in higher thannormal concentrations, are toxic to a human or animal. Generally, thecompositions according to the present invention comprise a vectorcomposition that is an admixture of a colloidal metal in combinationwith an agent which is toxic to a human or animal when the agent isfound in higher than normal concentration, or is in an unshielded formthat allows for greater activity than in a shielded form, or is found ina site where it is not normally found. When the vector composition isadministered to a human or animal, the agent is less harmful or lesstoxic or non-toxic to the human or animal than when the agent isprovided alone without the colloidal metal vector composition. Thecompositions optionally include a pharmaceutically-acceptable carrier,such as an aqueous solution, or excipients, buffers, antigenstabilizers, or sterilized carriers. Also, oils, such as paraffin oil,may optionally be included in the composition. The vector compositionsmay further comprise PEG or derivatives of PEG.

The compositions of the present invention can be used to vaccinate ahuman or animal against agents that are toxic when injected. Inaddition, the present invention can be used to treat certain diseaseswith cytokines or growth factors by administering the compositionscomprising agents such as cytokines or growth factors. By admixing theagents with the colloidal metal before administering the agents to thehuman or animal, the toxicity of the agent is reduced or eliminatedthereby allowing the factor to exert its therapeutic effect. Thecombination of a colloidal metal with such agents in a vectorcomposition reduces toxicity while maintaining or increasing thetherapeutic results thereby improving the efficacy as higherconcentrations of agents may be administered, or by allowing the use ofcombinations of different agents. The use of colloidal metals incombination with agents in vector compositions therefore allows the useof higher than normal concentrations of agents or administration ofagents that normally are unusable due to their toxicity, to beadministered to humans or animals. Preferably, the vector compositionsfurther comprise one or more types or sizes of PEG or derivatives ofPEG.

One embodiment of the present invention comprises methods for using avector composition comprising an agent associated with the colloidalmetal as a vaccine preparation. Among the many advantages of such avaccine is the reduction of toxicity of normally toxic agents. Thevector compositions used as a vaccine against agents may be prepared byany method. For example, the vector composition of an admixture ofagents and colloidal metal is preferably injected into an appropriateanimal. For example, rabbits weighing between approximately two to fivekilograms suffered no noticeable side-effects after they wereadministered, every two weeks, a composition comprising colloidal goldand the agent, 1 mg of cytokine, either IL-1 or IL-2. Because the agentis not toxic when administered according to the present invention, theoptimal quantity of the agent, which can function as an antigen, can beadministered to the animal. The vector compositions according to thepresent invention may be administered in a single dose or may beadministered in multiple doses, spaced over a suitable time scale.Multiple doses are useful in developing a secondary immunizationresponse. For example, antibody titers have been maintained byadministering boosters once a month.

The vaccine compositions may further comprise a pharmaceuticallyacceptable adjuvant, including, but not limited to Freund's completeadjuvant, Freund's incomplete adjuvant, lipopolysaccharide,monophosphoryl lipid A, muramyl dipeptide, liposomes containing lipid A,alum, muramyl tripeptide-phosphatidylethanoloamine, keyhole limpethemocyanin. A preferred adjuvant for animals is Freund's incompleteadjuvant and Alum for humans, which preferably is diluted 1:1 with thecompositions comprising a colloidal metal and an active agent.

A preferred method of use of the compositions of the present inventioncomprises administering to a human or animal an effective amount of avector composition comprising a colloidal metal admixed with at leastone agent, wherein the composition when administered to a human oranimal, is less or non-toxic, or has fewer or less severe side effectswhen compared to administration of the agent alone or in compositionswithout colloidal metals. The vector compositions according to thepresent invention can be administered as a vaccine against a normallytoxic substance or can be a therapeutic agent wherein the toxicity ofthe normally toxic agent is reduced thereby allowing the administrationof higher quantities of the agent over longer periods of time.

In practicing these embodiments, the route by which the composition isadministered is not considered critical. The routes that the compositionmay be administered according to this invention include known routes ofadministration, including, but are not limited to, subcutaneous,intramuscular, intraperitoneal, oral, and intravenous routes. Apreferred route of administration is intravenous. Another preferredroute of administration is intramuscular.

For example, it is known that Interleukin-2 (IL-2) displays significanttherapeutic results in the treatment of renal cancer. However, the toxicside effects of administration of IL-2 result in the death of asignificant number of the patients. In contrast, if a vector compositioncomprising at least IL-2 and a colloidal metal is administered, littleor no toxicity is observed and a strong immune response occurs in therecipient. The doses previously used for IL-2 therapy have been on theorder of 21×10⁶ units of IL-2 per 70 kg person per day (7×10⁶ units ofIL-2 per 70 kg person TID). One unit equals approximately 50 picograms,2 units equals approximately 0.1 nanograms, so 20×10⁶ units equals 1milligram. In one embodiment of this invention, the amount of IL-2 thathas been given to rabbits is approximately 1 mg per 3 kg rabbit. Ineffect, the studies of the effects of the administration of agentsdescribed herein have included doses of more than 20 times higher thanthat previously given to humans.

In another embodiment, where IL-2 (1 mg per 3 kg animal) wasadministered to 3 rabbits every third day for a two-week period, all theanimals appeared to be clinically sick, and two of the animals died fromthe apparent toxic effects of the IL-2. When the same dose of IL-2 wasused in vector compositions comprising colloidal gold and thenadministered to three rabbits for the same two-week period, no toxicitywas observed and a significant antibody response resulted in all threeanimals. A “positive antibody response” as used herein is defined as athree to fourfold increase in specific antibody reactivity, asdetermined by direct ELISA, comparing the post-immunization bleed withthe preimmunization bleed. A direct ELISA is done by binding IL-2 onto amicrotiter plate, and determining the quantity of IgG bound to the IL-2on the plate, by goat anti-rabbit IgG conjugated to alkalinephosphatase. Therefore, it is thought that the biological effects of theIL-2 remain. As the toxicity effects have been minimized, largerconcentrations of IL-2 may be administered if necessary where a larger,more effective immune response is required.

The present invention comprises methods for treating diseases byadministering vector compositions comprising one or more agents and acolloidal metal. The vector compositions may further comprise PEG orderivatives of PEG. It is theorized that after administration, theagents are released from the colloidal metal. Though not wishing to bebound by any theory, it is thought that the release is not simply afunction of the circulation time, but is controlled by equilibriumkinetics.

When a vector composition comprising at least a colloidal metal and atleast one agent was incubated with cells for 25 days, it was found thatonly 5% of the agent was released from the colloidal metal. Thus, it istheorized that circulation time alone does not explain the mechanismthrough which the agents are released from the complex in vivo. However,it has been found that the amount of agent released is, in part, relatedto the concentration of the complex in the body. When various dilutionsof compositions were analyzed (CytELISA™ assay system CytImmuneSciences, Inc.), it was found that the more dilute solutions of thecomplex released a significantly greater amount of agent. For example,there was essentially no release of agent in a 1:100 dilution of thecomplex, whereas over 35,000 pg. of the agent was released in a1:100,000 dilution of the composition.

Therefore, the lower the concentration of the composition in the largersolution, the greater the amount of agent released. The higher theconcentration of the composition, the lower the amount of agentreleased. Thus, it is theorized that due to the continuous in vivodilution of the compositions by blood and extracellular fluids, it ispossible to achieve the same therapeutic effect by administering a lowerdose of an agent to a patient than can be administered by previouslyknown methods.

It is also theorized that the amount of agent released from thecompositions of the present invention is related to the amount of agentinitially bound to the colloidal metal. More agent is released in vivofrom vector compositions having a greater amount of agents initiallybound. Thus, the skilled artisan could control the amount of agentsdelivered by varying the amount of agent initially bound to thecolloidal metal.

These combined properties provide methods by which a large amount ofagents can be bound to a colloidal metal, thereby rendering the agentless toxic than if administered alone. Then, a small amount of thevector composition can be administered to a patient resulting in theslow release of the agent from the complex. These methods provide anextended, low dose of the agents for the treatment of diseases such ascancer and immune diseases.

The compositions of the present invention are useful for the treatmentof a number of diseases including, but not limited to, cancer, bothsolid tumors as well as blood-borne cancers, such as leukemia;autoimmune diseases, such as rheumatoid arthritis; hormone deficiencydiseases, such as osteoporosis; hormone abnormalities due tohypersecretion, such as acromegaly; infectious diseases, such as septicshock; genetic diseases, such as enzyme deficiency diseases (e.g.,inability to metabolize phenylalanine resulting in phenylketanuria); andimmune deficiency diseases, such as AIDS.

Methods of the present invention comprise administration of the vectorcompositions in addition to currently used therapeutic treatmentregimens. Preferred methods comprise administering vector compositionsconcurrently with administration of therapeutic agents for treatment ofchronic and acute diseases, and particularly cancer treatment. Forexample, a vector composition comprising the agent, TNF, is administeredprior to, during or after chemotherapeutic treatments with knownanti-cancer agents such as antiangiogenic proteins such as endostatinand angiostatin, thalidomide, taxol, melphalan, paclitaxel, taxanes,vinblastin, vincristine, doxorubicin, acyclovir, cisplatin and tacrine.All currently known cancer treatment methods are contemplated in themethods of the present invention and the vector compositions may beadministered at different times in the treatment schedule as necessaryfor effective treatment of the cancer.

A preferred method comprises treatment of drug-resistant tumors, canceror neoplasms. These tumors are resistant to known anti-cancer drugs andtherapeutics and even with increasing dosages of such agents, there islittle or no effect on the size or growth of the tumor. Known in cancertreatment is the observation that exposure of such drug resistant tumorcells to TNF resensitizes these cells to the anti-cancer effect of thesechemotherapeutics. Evidence has been published that indicates that TNFsynergizes with topoisomerase II-targeted intercalative drugs such asdoxorubicin to restore doxorubicin tumor cell death. Also interferon(IFN) is known to synergize with 5-fluorouracil to increase thechemotherapeutic activity of 5-fluorouracil. The present invention canbe used to treat such drug-resistant tumors. A preferred methodcomprises administration of compositions comprising vectors having TNFand derivatized PEG bound to colloidal gold. With the pretreatment of apatient with a subclinical dose of TNF-cAu-PT, the tumor sequesters theTNF vector, sensitizing the cells to subsequent systemic chemotherapy.Such chemotherapies include, but is not limited to doxorubicin, otherintercalative chemotherapies, taxol, 5-fluorouracil, mitaxantrone,VM-16, etoposide, VM-26, teniposide, and other non-intercalativechemotherapies. Alternatively, another preferred method comprisesadministration of compositions comprising vectors having TNF and atleast one other agent effective for the treatment of cancer. Forexample, a PT-cAU_((TNF)) doxorubicin vector is administered to patientswho have drug resistant tumors or cancer. The amount administered isdependent on the tumor or tumors to be treated and the condition of thepatient. The vector composition allows for greater amounts of thechemotherapeutic agents to be administered and the vector also relievesthe drug-resistant characteristic of the tumor.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLES Example 1

Preparation of Colloidal Gold Sols

Colloidal gold is produced by the reduction of chloroauric acid (Au⁺³;HAuCl₄), to neutral gold (Au⁰) by agents such as sodium citrate. Themethod described by Horisberger, (1979) was adapted to produce 34 mmcolloidal gold particles. This method provided a simple and scalableprocedure for the production of colloidal gold. Briefly, a 4% goldchloride solution (23.03% stock; dmc², South Plainfield, N.J.) and a 1%sodium citrate solution (wt/wt; J.T. Baker Company; Paris, Ky.) weremade in de-ionized H₂O) (DIH₂O). 3.75 ml of the gold chloride solutionwas added to 1.5 L of DIH₂O. The solution was vigorously stirred andbrought to a rolling boil under reflux. The formation of 34 nm colloidalgold particles was initiated by the addition of 60 ml of sodium citrate.The solution was continuously boiled and stirred during the entireprocess of particle formation and growth as described below.

The addition of sodium citrate to the gold chloride initiated a seriesof reduction reactions characterized by changes in the color of theinitial gold chloride solution. With the addition of the sodium citratethe color of the gold chloride solution changed from a golden yellow toan intermediate color of black/blue. The completion of the reaction wassignaled by a final color change in the sol from blue/black to cherryred. After the final color change the solution was continuously stirredand boiled under reflux for an additional 45 minutes. Subsequently, thesol was cooled to room temperature and filter through a 0.22 u cellulosenitrate filter and stored at RT until use.

The formation of colloidal gold particles occurs in two stages:nucleation and particle growth. Particle nucleation was initiated by thereduction of Au⁺³ to Au⁰ by sodium citrate. This step is marked by acolor change of the gold chloride solution from bright yellow to black.The continuous layering of free Au⁺³ onto the Au⁰ nuclei drives thesecond stage, particle growth. Particle size is inversely related to theamount of citrate added to the gold chloride solution: increasing theamount of sodium citrate to a fixed amount of gold chloride results inthe formation of smaller particles, while reducing the amount of citrateadded to the gold solution results in the formation of relatively largerparticles.

Similar to the nucleation reaction, colloidal gold particle formation isalso correlated with a change in the solution's color. However, unlikethe initial reaction, this second color change is directly related toparticle size. When small particles (i.e., 12-17 nm) are made the sol isorange to red in color; when medium sized particles (i.e., 20-40 nm) aremade the sol appears red to burgundy in color and when large particles(i.e., 64-97 nm) are made the sols appear violet to brown in color.Critical to both particle nucleation and growth was the vigorousstirring of the reactants. Inadequate stirring at any step during theprocess resulted in the formation of heterogeneous particles with largerthan predicted diameters.

TEM (transmission electron microscopy) and dual angle light scatteringinterrogation of the colloidal gold preparations revealed that the sizeof the particles in the colloidal gold preparations were very close totheir theoretical size of 34 nm. The particles were homogenous in sizewith a mean particle diameter of 34-36 nm and a polydispersity measureaveraging 0.11 (Table IV). In this state the colloidal gold particlesstayed in suspension by their mutual electrostatic repulsion due to thenegative charge present on each particle's surface. Exposing these nakedparticles to salt solutions (i.e., NaCl at a 1% v/v final concentration)caused them to aggregate and ultimately precipitate out of solution.This process was blocked or inhibited by binding proteins (e.g., TNF) orother agents to the particles' surface.

Example 2

Metal Sources

Experiments were performed to see if the source of the starting goldreactants for the formation of the colloid formation affected thecolloidal gold compositions. Gold chloride was purchased from twodifferent commercial sources: Degussa Metals Catalysts Cerdec (dmc²) andSigma Chemical Company. Both gold preparations were analyzed for thepresence of contaminating metals as well as other substances. Theresults from these studies are listed in Table II. Although the goldconcentrations in each preparation were within reported values, it isclear that the Sigma preparation contains higher levels of Mg, Ca andFe.

TABLE II Purity of gold chloride salts used to generate colloidal goldElement dmc² Sigma Na <25 ppm >21 ppm Mg <25 ppm >60 ppm Ca <25 ppm >60ppm Fe <25 ppm >60 ppm

TEM of the particles revealed further differences between the particlesmade with different gold chloride sources, from Sigma and from dmc². Thecolloidal gold sols were manufactured as described above and observedused TEM. After cooling, 10 ml of the sol was centrifuged to concentratethe particles. The resultant supernatant was removed by aspiration andthe colloidal gold pellet was resuspended by gentle tituration. Thepellet was prepared for transmission electron microscopy followingstandard procedures.

The particles made with the Sigma gold chloride are translucent withapparent striations. The striations have been reported to be due to thepresence of trace contaminants, such as those identified above. Incontrast, the particles made with dmc² gold chloride are electron densewith very few striations.

Example 3

Generation of Colloidal Gold Sols Using Sigma and dmc² Gold Chloride

The above data suggested that the gold chloride from dmc² containedlower levels of contaminating elements. To determine the effect of thesetwo qualitatively different sources of gold chloride, colloidal goldsols were generated using the two different sources of the salt. Theprocedure for creating the colloidal gold particles follows theprocedure originally described by Horisberger, and in Example 1.Briefly, a 4% gold chloride (in water) solution was made from the dmc²and Sigma stock preparations. 3.75 ml of each solution was added toindividual flasks each containing 1.5 L of water. The solution wasbrought to a rolling boil, kept boiling under reflux, and vigorouslystirred. 22.5 ml of a 1% sodium citrate solution was added to eachflask. The solutions in both flasks were kept boiling until thewell-described process of colloidal gold formation was complete,signaled by a color conversion from gold to black to cherry red. Oncethe sols turned a cherry red, they were allowed to boil under reflux,with constant stirring, for an additional 45 min. After cooling, thesols were filtered through a 0.22 μm nitrocellulose filter and stored atroom temperature until use.

A qualitative comparison of the two sols was made with a standardlaboratory spectrophotometer, running a UV/VIS wavelength scan. Theresults revealed that the two batches of sols contained colloidal goldparticles with a similar mean diameter, as indicated by the wavelengthwhere the sol exhibits the greatest absorbance. However the moststriking difference between the two preparations is that the sol madewith the dmc² material had 3-times the number of particles as those madewith the Sigma material. In addition, it appeared that the distributionaround the lambda max is wider in the Sigma preparation than for thedmc² preparation, indicating that the particles generated with the Sigmasalt are more heterogeneous than those generated with the dmc² goldchloride.

Example 4

Analytical Comparison of the Colloidal Gold Sols

The above qualitative differences were confirmed by quantitativeparticle characterization with a Brookhaven Particle Sizer. For thesestudies the samples of particles from each gold source were preparedaccording to manufacture's instruction. The data are presented below inTables II and III. The data confirmed that the particles in bothpreparations are of approximately the same size (34-37 nm). Neverthelessthe sols made with the dmc² material have a 3-fold higher particledensity than those made with the Sigma material. In addition, theparticles made with the Sigma gold chloride preparation are 2.5 timesmore heterogeneous (i.e., have a larger value for their polydispersity)than the particles made with the dmc² material (Table IV).

TABLE III Variable wavelength analysis of colloidal gold sols generatedwith dmc² and Sigma gold chloride Sample λ Max Absorbance λ@ ½ Max dmc²526 nm 2.8899 576 Sigma 529 nm 1.0513 587

TABLE IV Mean particle size and distribution of colloidal gold solsgenerated with dmc² and Sigma gold chloride Particle Size MeanPolydispersity dmc² 34.0 0.096 Sigma 36.9 0.230

Example 5

Determination of the pH Binding Optimum

The binding of proteins to colloidal gold is known to be dependent onthe pH of the colloid gold and protein solutions. The pH binding optimumof TNF to colloidal gold sols was empirically determined. This pHoptimum was defined as the pH that allowed TNF to bind to the colloidalgold particle, but blocked salt-induced (by NaCl) precipitation of theparticles. Naked colloidal gold particles are kept in suspension bytheir mutual electrostatic repulsion generated by a net negative chargeon their surface. The cations present in a salt solution cause thenegatively charged colloidal gold particles, which normally repel eachother, to draw together. This aggregation/precipitation is marked by avisual change in the color of the colloidal gold solution from red topurple (as the particles draw together) and ultimately black, when theparticles form large aggregates that ultimately fall out of solution.The binding of proteins or other stabilizing agents to the particles'surface will block this salt-induced precipitation of the colloidal goldparticles.

The pH optimum of TNF binding to colloidal gold was determined using 2ml aliquots of 34 nm colloidal gold sol whose pH was adjusted from pH 5to 11 (determined by using pH strips) with 1N NaOH. TNF (KnollPharmaceuticals; purified to homogeneity) was reconstituted in diH₂O toa concentration of 1 mg/ml and further diluted to 100 μg/ml in 3 mM TRISbase. To determine the pH binding optimum for TNF, 100 μl of the 100μg/ml TNF stock was added to the various aliquots of pH-adjustedcolloidal gold. The TNF was incubated with the colloid for 15 minutes.Subsequently 100 μl of a 10% NaCl solution was added to each of thealiquots to induce particle precipitation. The optimal binding pH wasdefined as the pH, which allowed TNF to bind to the colloidal goldparticles, while preventing the particles' precipitation by salt.

Example 6

Saturation Binding Studies

Based on the data obtained from the pH binding study, the pH of 34 nmcolloidal gold sol was adjusted to pH 8 with 1 N NaOH. The sol wasdivided into 1 ml aliquots to which increasing amounts (0.5 to 4 μg ofTNF) of a 100 μg of TNF/ml solution were added. After binding for 15minutes the samples were centrifuged at 7,500 rpm for 15 minutes. A 10μl sample of the supernatant was added to 990 μl of EIA assay diluent(provided as part of a commercial EIA kit for TNF measurement; CytlmmuneSciences, Inc., College Park, Md.). The remainder of the supernatant wasremoved by aspiration and the colloidal gold pellet was resuspended toits original volume in a PEG 1450/diH₂O solution pH 8. 10 μl of theresuspended pellet was added to 990 μl of EIA assay diluent. Thereconstituted pellet and supernatant solutions were serially diluted andanalyzed for TNF concentration by a quantitative commercial EIA(Cytlmmune Sciences, Inc, College Park, Md.).

Using the data from the pH binding study, the pH of 50 ml of colloidalgold was adjusted between 8.0-9.0. At this pH the binding of TNF to afixed volume of colloidal gold exhibited saturation kinetics (FIG. 2).As shown in FIG. 2, at 0.5 μg of TNF/ml of gold sol virtually all theTNF was bound to the colloidal gold particles with an insignificantamount (2-5%) present as free TNF in the supernatant. This colloidalgold-TNF complex precipitated in the presence of salt, indicating thatthis concentration of TNF did not fully coat the colloidal goldparticles and is a sub-saturating dose of TNF. By increasing the TNFconcentration the amount of TNF bound to the colloidal gold particlesprogressively increased with relatively little change in the amount offree TNF measured in the supernatant. This increase in particle-boundTNF paralleled the increase in the particles' stability againstsalt-induced precipitation. Saturation of the colloidal gold particleswith TNF occurred when all the binding sites on the surface of theparticles were bound with TNF. Saturation of the colloidal goldparticles occurred at a binding concentration of 4 μg/ml (FIG. 2).Binding at doses above 4 μg/ml resulted in increasing amounts of freeTNF measured in the supernatant.

In FIG. 2, saturation binding of TNF to colloidal gold is shown. 50 mlof 34 nm colloidal gold sol was pH adjusted to 8 using 1 NaOH, and thendivided into 1 ml aliquots. Increasing volumes of a stock TNF (100 μg/mlin 3 mM TRIS) solution were added to the aliquots and allowed to bindfor 15 minutes. The samples were centrifuged at 7500 rpms for 15minutes. A 10 μl sample of the supernatant was diluted in a Trisbuffered saline milk solution (assay diluent). The remainder of thesupernatant was removed by aspiration and the colloidal gold pellet wasresuspended by gentle tituration. 10 μl of the resuspended pellet wasdiluted in assay diluent. Both pellet and supernatant samples wereserially diluted and measured for TNF concentration by EIA (CytImmuneSciennces, Inc.).

Example 7

Large Scale Production of the Various Colloidal Gold Vectors

The in vivo assessment of the colloidal gold-TNF particles, alsoreferred to as vectors, required the scaling-up of all manufacturingprocedures. Large (8 L) batches of colloidal gold were manufactured asdescribed above. The manufacturing procedure of the colloidal gold solswas adapted to 8 L colloidal gold production. A reflux apparatus (KontesGlass, Vineland, N.J.) was used to generate 8 L of colloidal gold for invivo experiments. Briefly, 8 L of diH₂O was heated to a rolling boil. 20ml of gold chloride was added through one port, followed by the additionof 320 ml of sodium citrate. The color change of the resultant sol wasthe same as that seen in the smaller preparation. Once the cherry redcolor was achieved the sol was allowed to cool overnight, and was thensterile filtered as described above.

The particles made using large scale amounts were essentially the sameas particles made using bench scale methods. See Table V.

TABLE V Characterization of bench scale and large scale preparations of34 nm colloidal gold sols by dynamic light scattering. Measured SizePreparation nm Polydispersity Bench Scale/1.5 L 36 0.131 LargeScale/(8.0 L) 34 0.096

Next, the uniform coating of the colloid particles had to beaccomplished. This was an important consideration since analysisdemonstrated that the binding between the colloidal gold particles andthe TNF molecules was nearly instantaneous. Consequently, simply addinga concentrated protein solution to a large volume of gold would resultin particles that were differentially coated with TNF. To optimize theinteraction of the particles and TNF molecules, apparatus that allowedcomplete interaction between the colloidal gold sol and the TNF solutionwas used. A schematic representation of the apparatus is shown inFIG. 1. The apparatus reduced the mixing volume between the nakedcolloidal gold particles and TNF by drawing each component into a smallmixing chamber (a T-connector). The colloidal gold particles and the TNFsolutions were physically drawn into the T-connector by a singleperistaltic pump that drew the colloidal gold particles and the TNFprotein from two large reservoirs. To further ensure proper mixing, anin-line mixer (Cole-Palmer Instrument Co., Vernon Hills, Ill.) wasplaced immediately downstream of the T-connector. The mixer vigorouslymixed the colloidal gold particles with TNF, both of which were flowingthrough the connector at a flow rate of approximately 1 L/min.

Prior to mixing, the pH of the gold sol was adjusted to pH 8 using 1 NNaOH, while the recombinant human TNF was reconstituted and prepared in3 mM Tris. The solutions were added to their respective sterilereservoirs using a sterile closed tubing system. Equal volumes of thecolloidal gold sol and the TNF solution were added to the appropriatereservoirs. Since the gold and TNF solutions were mixed in equalvolumes, the initial starting TNF concentration for each test vector wasdouble the final concentration. For example, to make 4 L of a 0.5 μg/mlsolution of cAu-TNF, 2 L of colloidal gold were placed in the goldreservoir, while 2 L of a 1 μg/ml TNF solution was added to the TNFcontainer.

Once the solutions were properly loaded into their reservoirs, theperistaltic pump was activated, drawing the TNF and the colloidal goldsolutions into the T-connector, through the in-line mixer, theperistaltic pump, and into a large collection flask. The resultantmixture was stirred in the collection flask for 15 minutes. After thisbinding step, 1 ml samples from each of the formulations were collectedand tested for salt precipitation. A 1.0 μg/ml and a 4.0 μg/mlpreparations were processed as described below, while a third solution,a second 0.5 μg/ml preparation, was treated by adding niPEG-thiol 5,000(10% v/v addition of a 150 μg/ml stock in diH₂O) at a finalconcentration of 15 μg/ml. This third solution, a PEG-thiol-colloidalgold-TNF (PT-cAu-TNF) solution, was incubated for an additional 15minutes. Two other PT-cAu-TNF formulations were made using a 20,000 anda 30,000 MW form of PEG-Thiol. During these studies additional controlswere tested for comparison including PEG-Thiol/naked colloidal gold orthe 4 μg/ml cAu-TNF vector.

Colloidal gold bound TNF in each preparation was separated from free TNFby diafiltration through a 50,000 MWCO BIOMAX diafiltration cartridge(Millipore Corporation, Chicago, Ill.). An aliquot of the permeate(i.e., free TNF) was removed and set aside for TNF determination. Formass balance determination, the total volume of the permeate wasmeasured. The retentate, which contained the TNF bound colloidal gold,was sterile filtered through a 0.22 micron filter and a 10 μl aliquotwas taken for TNF analysis. The remainder of the retentate was frozen at−80° C. for storage. Subsequent to the determination of the TNFconcentrations, a solution of native TNF was manufactured in 3 mM Trisand used as the control for the in vivo studies.

Example 8

Initial Formulation of the Colloidal Gold TNF Vector

This series of experiments was designed to determine the effect ofvarious TNF:colloidal gold binding ratios on the in vivo biologicactivity of the colloidal gold TNF vector. Three different formulationsof the colloidal gold TNF vector were made based on the data generatedfrom the TNF-binding-to-colloidal-gold saturation curve. The threevectors were generated by binding TNF at 1, 2 or 4 μg of TNF/ml ofcolloidal gold solution. These three vectors differed in their abilityto remain colloidal following the addition of salt. The 1 μg/ml vectorprecipitated immediately (i.e., the color of the colloidal changed fromcherry red to black) upon the addition of the salt solution. In contrastthe color of the 2 μg/ml vector turned from red to purple, indicating anaggregation of the colloidal gold particles. Finally, the 4 μg/mlpreparation remained red after the addition of salt, indicating that theparticles remained colloidal and did not interact. Although thecolloidal nature of the particles in the 1 and 2 μg/ml vectors wasaltered by their exposure to salt, they remained stable when incubatedwith normal human plasma. These data suggested that plasma factors, mostlikely blood borne proteins, bound to the particle and immediatelystabilized it against precipitation. Thus exposing these vectors toblood prevented their precipitation and allowed for their investigationin vivo.

Comparative safety studies of the three cAu-TNF vectors and native TNFwere done in MC-38 tumor-burdened C57/BL6 mice. The toxicity profile ofnative TNF was dose-dependent. 5 μg of native TNF/mouse causedpiloerection and diarrhea within 1-2 hours of injection. With increasingdoses of native TNF more severe toxicities were observed. At a dose of15 μg of TNF/mouse, 50% of the animals became hypothermic andunresponsive, and ultimately died within 24 hours. The mice were scoredat different times after injection using the following toxicity ratingscale: 0=normal activity; 1=piloerection; 2=loose stools; 3=lethargy;4=unresponsive; and 5=death.

Although these three cAu-TNF vectors were biologically similar to nativeTNF preparations in the in vitro bioassay, their toxicity profiles werequite different in the C57/BL6-MC-38 tumor model. Increasing the initialbinding concentration of TNF from 1.0 to 4.0 μg/ml increased therelative safety of the cAu-TNF vector (FIG. 3A). Mice injected with 15μg of native TNF had a 50% mortality rate. A 15 μg injection of the 1.0μg/ml cAu-TNF vector also caused a 50% mortality rate. In contrast, micereceiving 15 μg of the cAu-TNF bound at 2.0 μg/ml had a reducedmortality rate of 25%. Finally, none of the mice injected with 15 μg ofthe 4.0 μg/ml cAu-TNF preparation died. This last group of animalsexhibited only transient toxicities that resolved within 8 hours oftreatment.

FIG. 3A shows the effect of TNF:gold binding ratios on the safety of thecAu-TNF vector. Three different colloidal gold TNF vectors weregenerated based on their relative degree of TNF saturation of thecolloidal gold particles. MC-38 tumor burdened C57/BL6 mice (n=4/group)were intravenously injected with 15 μg of either native TNF, not boundto a gold vector, or one of the three cAu-TNF vectors. The mice werescored at various points after the injection using the toxicity ratingscale described. The percent survival for the treatments were: NativeTNF=50%, 1 μg/ml cAu-TNF vector=25%, 2 μg/ml cAu-TNF vector=75% and 4μg/ml cAu-TNF vector=100%.

A second dose escalation and safety study with the 4.0 μg/ml cAu-TNFvector indicated that this composition was safer on a dose-to-dose basiswhen compared to native TNF (FIG. 3B). Treatment with 12 μg or 24 μg ofthis cAu-TNF vector per mouse resulted in significant tumor reduction(FIG. 3C). In effect, this cAu-TNF vector increased the relative safetyof any given dose of TNF and at a maximally tolerated dose improved thetreatment's efficacy. These safety and efficacy data suggested that thiscAu-TNF vector effectively increased the therapeutic index for TNF,since the drug's efficacy was maintained while its safety was improved.

FIG. 3B shows the dose escalation and toxicity of native TNF and 4 μg/mlcAu-TNF in MC-38 tumor-burdened C57/BL6 mice. MC-38-tumored C57/BL6 mice(n=4/group/dose) were intravenously injected with increasing doses ofnative TNF or the 4 ug/ml cAu-TNF vector. Mice were scored using thetoxicity rating scale described. The percent of the animals survivingnative TNF treatment at 6, 12, and 24 μg/mouse were 100%, 75% and 0%respectively. All animals receiving the cAu-TNF treatment survived.*p≦0.05.

FIG. 3C shows a comparison of the anti-tumor efficacy of native TNF andthe 4 μg/ml cAu-TNF vector in MC-38 tumor-burdened C57/BL6 mice. Theanti-tumor responses for the various treatment groups described in FIG.3B were measured by determining three dimensional (L×W×H) tumormeasurements 10 days after treatment. Data are presented as the mean±SEMof tumor volume (in cm³) for the various groups. All animals receivingthe 24 μg native TNF treatment died within 24 hours of treatment.*p≦0.05 versus untreated controls.

These data strongly suggested a preferred composition for the cAu-TNFvector, which was then tested for its biodistribution. Over time, thebiodistribution of TNF was different between those animals treated withnative TNF and those treated with cAu-TNF. One hour after injection,mice receiving native TNF had higher levels of TNF in the kidneycompared to cAu-TNF treated mice (FIG. 3D). In contrast, eight hoursafter injection, mice receiving the colloidal gold formulation hadhigher levels of TNF in the tumor (FIG. 3E). Thus it seemed that thecAu-TNF vector was improving safety and maintaining efficacy bytargeting the delivery of TNF to the tumor.

FIGS. 3D and 3E show the comparison of the TNF distribution profiles inMC-38 tumor burdened C57/BL6 mice intravenously injected with 15 μgnative TNF or the 4 μg/ml cAu-TNF vector. MC-38 tumor-burdened C57/BL6mice (n=4/group/treatment/time point) were intravenously injected with15 μg of native TNF or the 4 μg/ml cAu-TNF vector. A group of animalswere sacrificed either 1 (FIG. 3D) or 8 hours (FIG. 3E) after injectionand organs were collected. The organs were flash frozen at −80° C. andstored until analyzed. The organs were quickly defrosted by addition 1ml of PBS (containing 1 mg/ml of bacitracin and PMSF) and homogenizedusing a polytron tissue disrupter. The homogenate was centrifuged at5000 rpms and the resultant supernatant was analyzed for TNFconcentration and total protein as described above. Data are presentedas the mean ±SEM from four organs per time point.

Autopsy of the animals revealed a potential problem with this cAu-TNFvector. The dramatic black color of the liver and the spleen of cAu-TNFvector treated mice argued that part of the improved safety may havebeen due to the vector's uptake and clearance by these organs. Furtherstudies revealed that this uptake was rapid, often occurring within 5minutes after intravenous injection. Visual inspection of these organssuggested that the black color of these organs was not different fromthe black precipitates formed when naked colloidal gold particles wereexposed to salt. Also, it is unlikely that the black color of theseorgans was due to trapped blood in these organs since the animals wereheparinized and extensively perfused prior to organ collection. Thesedata suggested that a majority of the vector was rapidly cleared by thecomponents of the RES, leading to the conclusion that the cAu-TNF vectorwas not optimized.

Additional evidence supporting this hypothesis was derived frompharmacokinetic studies that compared TNF levels between native TNF andtwo cAu-TNF vectors. Contrary to expectations, administration of the 4ug/ml cAu-TNF vector resulted in lower initial serum levels than nativeTNF (FIG. 3F). The TNF levels of those mice given the cAu-TNF vectorwere consistently 2-5 fold lower than that measured in animals receivingnative TNF. This difference in PK was more pronounced with a 0.5 μg/mlcAu-TNF vector, where the serum levels of TNF were nearly 10-fold lowerthan that seen with the native preparation. Mice receiving cAu-TNFvectors, regardless of the formulation (i.e., 0.5 or 4.0 μg/ml),consistently exhibited lower blood levels of TNF than mice receiving thenative protein. Taken together, the vector biodistribution and PK dataargued that uptake by the RES needed to be significantly reduced oreliminated for this vector to effectively target the tumor.

FIG. 3F shows a comparison of the pharmacokinetic profiles of native TNFor the 4 ug/ml cAu-TNF vector in MC38-tumor burdened C57/BL6 mice. MC-38tumor burdened mice (n=3/group/time point) were intravenously injectedwith 10 μg of either native TNF or the 4 ug/ml cAu-TNF vector. At theindicated time points the mice were anesthetized and bled through theretro-orbital sinus. The blood samples were allowed to clot and werecentrifuged at 14,000 rmps. The resultant sera samples were analyzed forTNF concentration using a commercial EIA for TNF. Data are presented asthe mean±SEM serum concentration from three mice per time point.*p<0.05.

Example 9

Vectors to Avoid Clearance by the RES and Target the Delivery of TNF toSolid Tumors

Recognition and clearance of foreign objects by the RES has been seenwith other drug carriers. For liposomes and biodegradable polymers thisproblem was addressed by surface modifications using a variety of PEGstabilizers as well as block co-polymers, such as polaxamer andpolaxamine. Numerous stabilizers, including those used in liposomeformulations (e.g., carbowax 20M, tetronic 407, pluronic 908) were addedto the 4.0 ug/ml cAu-TNF vector. None of the reagents effectivelyblocked the vector's uptake by the RES.

Next, the amount of TNF bound per particle was reduced. TNF was firstbound to the colloidal gold particles at a sub-saturating dose (i.e.,0.5 μg/ml). Thiol-derivatized polyethylene glycol (PEG-Thiol; MW=5,000)was then added to the particles. This small, linear PEG-Thiol reagentwas chosen because thiol groups could bind directly to the particle'ssurface, presumably in between the molecules of TNF. This new vector wastested in the MC-38 tumor burdened C57/BL6 mice.

This composition of the colloidal gold bound TNF vector was formulatedby binding TNF and an additional agent to the same particle of colloidalgold. This vector was formed by first binding TNF to colloidal gold at asub saturating dose of 0.5 μg/ml. A derivatized PEG was then added tothe vector. The derivatized PEG was a thiol-derivatized polyethlyleneglycol (methoxy PEG-Thiol, MW: 5000 dalton, PEG-Thiol, Shearwater Corp.,Huntsville, Ala.). The final concentration of PEG-Thiol was 15 μg/ml,which was added as a 10× concentrate in diH₂O. Thiol-derivatized PEGsare good components for colloidal gold vectors since the thiol groupbinds directly to the surface of the colloidal gold particles. A 5,000MW thiol-PEG was the first thiol-derivatized PEG to be tested.Additionally, efficacy experiments using mPEG-thiol with MWs of 20,000and 30,000 daltons were performed as described below.

The biodistribution profile seen following the administration of thePEG-Thiol modified cAu-TNF (PT-cAu-TNF) vector was different from thoseobserved with the previous cAu-TNF vectors. With this new vector, theliver and spleen did not visibly take up the PT-cAu-TNF vector, (FIG.4A) as occurred with the 0.5 and 4.0 μg/ml cAu-TNF vectors (FIG. 4B).FIG. 4C shows an untreated liver and spleen. As striking as theinhibition of the RES uptake, was the apparent accumulation of thePT-cAu-TNF vector in the MC-38 tumor, since the tumors acquired thebright red/purple color of the colloidal gold particle within 30-60minutes of vector's administration. The sequestration continuedthroughout the time course of the study and was coincident with theaccumulation of TNF in the tumor and extended blood residence time ofTNF.

Unlike the black color of the gold that accumulated in the liver andspleen following the 0.5 μg/ml and 4.0 μg/ml cAu-TNF vector treatment,the color of the gold observed accumulating in the tumor following theadministration of the PT-cAu-TNF was reddish-purple. This difference issignificant because it indicates that the gold particles remained in acolloidal state during their residence in the circulation and theiraccumulation in the tumor. Interestingly, the pattern of PT-cAu-TNFaccumulation in and around the tumor site changed with time. ThePT-cAu-TNF was initially (i.e., 0-2 hours) sequestered solely in thetumor. With time, vector staining was apparent on the skin and on thesurrounding ventral tissues of the mouse. During the blunt dissection ofsacrificed animal's tumor, it was observed that the extra-tumor stainingin these animals was restricted to the dermal layer where the tumorcells were initially implanted. Minimal staining was present on theunderlying muscle bed on which the tumor rested. This observationsuggested that the peripheral staining may be due to the accumulation ofthe vector in the blood vessels, possibly new blood vessels, feeding thetumor mass. Currently, it is unknown whether the staining represents anactive sequestration of the drug in these blood vessels or the passiveaccumulation due to tumor saturation with the vector.

To determine whether the staining reflected a hemorrhagic response causeby TNF, the staining pattern mice receiving a 15 μg injection of the 4ug/ml cAu-TNF vector or native TNF was compared with those receiving thesame dose of the PT-cAu-TNF vector. Mice treated with the 4 ug/mlcAu-TNF vector began to exhibit the tumor scar formation which typicallyfollows intravenous administration of TNF. A similar pattern of scarringwas observed following native TNF treatment. The pattern of the scarstaining observed with the native TNF or the 4 ug/ml cAu-TNF vectortreatments was clearly distinct from the pattern of staining observedfollowing PT-cAu-TNF vector treatment. Further evidence that thestaining pattern observed following the administration of the PT-cAu-TNFvector was obtained from mice receiving PEG-Thiol colloidal goldparticles initially bound with murine serum albumin (MSA). ThePT-cAu-MSA vector caused staining of the tumor like that of thePT-cAu-TNF vector albeit at a much slower rate. The staining of thetumor was similar in color to that observed with PT-cAu-TNF treatment.However the change in tumor coloration was only evident after 4 hours oftreatment, compared with the 30-60 minute color change observed with thePT-cAu-TNF vector. Furthermore, the intensity of the staining was lowerthan that observed with the PT-cAu-TNF vector.

FIG. 4 shows inhibition of the RES-mediated uptake of the colloidal goldTNF vector by PEG-Thiol vectors. The PT-cAu-TNF vector was developedusing specified ratios of TNF and PEG-Thiol as described. After binding,the vector was concentrated by diafiltration and analyzed for TNFconcentration by EIA. 15 μg of the PT-cAu-TNF vector was intravenouslyinjected into MC-38 tumor-burdened C57/BL6 mice. The mice weresacrificed 5 hours after the injection and perfused with heparinizedsaline. The livers (on left of picture) and spleens were photographed.

Example 10

Pharmacokinetic and Distribution Analyses

In general, these experiments comprise native TNF, cAu-TNF (4 μg/ml), orPT-cAu-TNF, (0.5 μg/ml) vectors that were generated as described above.Depending on the study, 5-20 μg of native or one of the cAu-TNF vectorswere intravenously injected, through the tail vein, of MC-38tumor-burdened mice. Mice were bled at 5, 180, and 360 minutes afterinjection through the retro-orbital sinus. The blood was allowed to clotand the resultant serum was collected and frozen at −20° C. for batchTNF analysis by EIA (Cytlmmune Sciences, Inc.). At selected time points,various organs were collected and flash frozen. To determine TNFcontent, the organs were defrosted, homogenized, and centrifuged at14,000 rpms for 15 minutes. The supernatant was analyzed for TNFconcentration as described above, as well as for total protein bydetermining the sample's absorbance a 280 nm. Organ TNF concentrationswere normalized to total protein.

A. Gold Distribution

Various organs, including liver, lung, spleen, brain, and blood, wereexamined for the presence of elemental gold following the intravenousinjection of 15 μg of the PEG-Thiol stabilized 0.5 μg/ml cAu-TNF vector.The mice were sacrifice 6 hours the injection; blood was collected andthe various organs harvested, including liver, spleen, and tumor. Afterremoval, the organs were digested in aqua-regia (3 parts concentratedHCl to 1 part concentrated nitric acid) to extract the gold present inthese organs. The extraction was carried out over 24 hours, after which,the samples were centrifuged at 3500 rpms for 30 minutes. Thesupernatants were analyzed for the presence of total organ goldconcentration by inductively coupled plasma spectroscopy. The resultsare reported as total organ gold concentration (in ppm) in FIG. 5A. Theresults demonstrate that the intra-tumor concentration of gold wasnearly 2-fold higher than that measured in liver and nearly 7-foldhigher than that found in the spleen. Although this pattern suggeststhat the vector was retained in the tumor compared to other organs, weobserved that the highest level of gold was still in the circulation ofthese animals.

In FIG. 5A, gold distribution in various organs of MC-38 tumor burdenedC57/BL6 mice is shown. The supernatants were analyzed for the presenceof total organ gold concentration by inductively coupled plasmaspectroscopy. The results are reported as total organ gold concentration(in ppm) for 3 mice per organ. *p<.0.05 versus liver and spleen; \p<0.05 versus spleen.

B. Distribution and Pharmacokinetics of TNF

The sequestration of colloidal gold within the tumor mass was paralleledby the prolonged presence of the drug vector in the circulation as wellas the active accumulation of TNF in the tumor. Unlike the cAu-TNFvectors without derivatized PEG, injection of the PT-cAu-TNF vectorresulted in elevated levels of TNF in the circulation throughout thetime course studied. Six hours after injection with native TNF, the TNFlevels were only 2% of that seen at 5 minutes (FIG. 5B). In contrast,mice receiving the PT-cAu-TNF vector had TNF blood levels which wereapproximately 30% of their maximal 5-minute values. At this 6-hour timepoint, blood TNF levels in mice treated with the PT-cAu-TNF formulationwere 23-fold higher than those in mice treated with native TNF.

FIG. 5B shows the TNF pharmacokinetic analysis. Mice were bled throughthe retro-orbital sinus at 5, 180 and 360 minutes after the injection.The blood samples were centrifuged at 14,000 rpms and the resultantserum analyzed for TNF concentration using an EIA. Data are presented asthe mean±SEM serum TNF concentration from 3 mice/time point. (*p<0.05).

In those animals treated with PT-cAu-TNF vector, TNF accumulated in thetumor. As shown in FIG. 5C, the maximal intra-tumor concentration of TNFobserved in those mice treated with native TNF was 0.8 ng of TNF/mgprotein. The peak amount was seen within five minutes of administrationof the native TNF, and did not increase over the 6-hours. In contrast,those animals treated with PT-cAu-TNF vector had intra-tumor levels ofTNF that increased over time. TNF was actively sequestered in the tumorof those animals treated with PT-cAu-TNF vector. By the end of the timeperiod, nearly 10-times more TNF was found in the tumors of thoseanimals treated with the PT-cAu-TNF vector compared to those treatedwith native TNF or the 4 μg/ml cAu-TNF vector (FIG. 5D).

In FIG. 5C, the intra-tumor TNF distribution over time is shown. Micewere sacrificed 5, 180 and 360 minutes after the injection of 15 μg ofnative TNF or PT-cAu-TNF vector. The tumors were removed and analyzedfor TNF and total protein. Data are presented as the mean±SEM of tumorTNF concentration, expressed in ng TNF/mg of total protein, from 3mice/time point/treatment group. (Δp<0.1, *p<0.05).

FIG. 5D shows a comparison of the intra-tumor TNF concentrations fromanimals injected intravenously with 15 μg of either the 4 μg/ml cAu-TNFvector or the PT-cAu-TNF vector.

The accumulation of the PT-cAu-TNF vector in the MC-38 tumor mass wasnot a passive event or just a function of the vector's extendedresidency time in the circulation since, over the same period of time,TNF did not accumulate in other organs, such as the lung, liver, andbrain. Rather, the presence of TNF in these organs was similar inpattern to that seen in blood. Furthermore, the distribution of the drugin these non-targeted organs was similar to that seen with native TNF.Consequently, the accumulation of TNF resulting from the administrationof the PT-cAu-TNF vector is specific to the tumor (FIGS. 5E and F).

FIGS. 5E and F show the distribution of TNF in various organs from MC-38tumor-burdened C57/BL6 mice receiving either native TNF (FIG. 5E) orPT-cAu-TNF (FIG. 5F). Livers, lung and brains from the animals treatedin this Example were processed and analyzed for TNF and proteinconcentrations. Data are presented as the mean±SEM of intra-organ TNFconcentration from 3 mice/time point/formulation injected.

Example 11

Dose Escalation, Toxicity and Efficacy

C57/BL6 mice were implanted with the colon carcinoma cell line, MC-38,as a model to compare the safety and efficacy of native and colloidalgold bound TNF preparations. C57/BL6 mice were implanted with 10⁵ MC-38tumor cells in one site on the ventral surface. The cells were allowedto grow until they formed a tumor measuring 0.5 cm as determined bymeasuring the tumor in three dimensions (L×W×H). MC-38 tumor burdenedC57/BL6 mice (n=4-9/group) were intravenously injected with increasingdoses of native TNF, cAu-TNF vector, or PT-cAu-TNF vector. The mice weredivided into nine groups with 4-9 animals/group. One group served as anuntreated control group. Two groups were intravenously injected witheither 7.5 or 15 μg of native TNF, (FIG. 6A). Two groups wereintravenously injected with either 7.5 or 15 μg of a 20K-PT-cAu-TNFvector (FIG. 6B). Two groups were intravenously injected with either 7.5or 15 μg of the 30K-PT-cAu-TNF vector (FIG. 6C). Tumor measurements weremade on various days after the treatment on animals that survived TNFtreatment. Statistical difference between the various groups wasdetermined using a paired t-test.

The mice were scored at different times after injection using thefollowing toxicity rating scale: 0=normal activity; 1=piloerection;2=loose stools; 3=lethargy; 4=unresponsive; and 5=death. Animals scoringa 4 on two consecutive scoring times were sacrificed. Treatment efficacywas determined by monitoring the reduction in tumor volume induced bythe various TNF treatments. The measures were compared to the initialtumor volume of each animal in the various groups as well as animalsreceiving saline injections (untreated controls). Untreated controlswere sacrificed when their tumor volumes were 4 cm³.

A 5K-PT-cAu-TNF vector, comprising PEG-thiol of 5,000 mw, was tested forsafety and efficacy in dose escalation studies in MC-38 tumor-burdenedmice. Like the 4 ug/ml cAu-TNF vector, the 5K-PT-cAu-TNF vector had animproved safety profile when compared to native TNF. At a dose of 15 μgof native TNF/mouse, 33% (3 out of 9) of the animals died within 24hours of treatment. In addition, 7.5 μg of native TNF resulted in 1 outof the 9 animals dying. In contrast, none of the animals receivingeither 7.5 or 15 μg of TNF bound to the 5K-PT-cAu-TNF vector died. Thesevector-treated animals exhibited only transient adverse clinicaleffects. No appreciable difference in the anti-tumor efficacy of the 5KPT-cAu-TNF vector, compared to native TNF, was observed following asingle treatment (FIG. 6A). These findings were replicated in twoexperiments.

FIG. 6A is a graph comparing safety and efficacy of native TNF orPT-cAu-TNF vectors. \ p<0.05 for the 7.5 μg of dose of native TNF orPT-cAu-TNF treatment versus untreated controls. *p<0.05 for the 15 μg ofdose of native or PT-cAu-TNF treatment versus untreated controls and 7.5μg of dose native and PT-cAu-TNF.

The effect of PEG-thiol chain length on vector anti-tumor efficacy isshown in FIGS. 6B-C. At the highest TNF dose (15 μg) no differences werenoted among any of the TNF vectors compared to native TNF. However, at alower dose of 7.5 μg of TNF, a pattern emerged. As noted above, 5KPEG-thiol did not markedly improve the anti-tumor effect of thecolloidal gold vector compared to native TNF. By increasing the PEGchain length to 20K there was a slight, non-statistical improvement intumor reduction (FIG. 6B), while increasing it to 30K resulted in amarked, statistically significant improvement in tumor regression (FIG.6C). With the 30K PT-cAu-TNF vector, animals treated with a dose of 7.5μg of TNF via this vector, had residual tumors of similar size to thosetreated with 15 μg native TNF. In contrast to those treated with 15 μgof native TNF, the 30K PT-cAu-TNF-treated animals administered 7.5 μg ofTNF experience no toxicity. In effect, a single injection of a 30KPT-cAu-TNF vector which gave less TNF but induced the same maximalanti-tumor regression as that seen with twice as much native TNF, andthe treated subject survived the treatment. In this tumor model, asingle injection of either 5K or 20K PEG-Thiol-cAu-TNF vector was saferthan native TNF, and the 30K PEG-Thiol-cAu-TNF vector was both safer andmore efficacious than the native molecule.

FIG. 6B is a graph comparing native TNF and 20K-PT-cAU-TNF safety andefficacy. †,§ p<0.05 for the 7.5 μg of dose of native TNF or PT-cAu-TNFvector treatment, respectively, versus untreated controls. 7.5 μg of the20 K-PT-cAu-TNF vector was not statistically different form the 7.5 μgnative group *p<0.05 for the 15 μg of dose of native or PT-cAu-TNFtreatment versus untreated controls and the 7.5 μg of native and20K-PT-cAu-TNF vector.

FIG. 6C is a graph comparing native TNF and 30K-PT-cAU-TNF safety andefficacy. \ p<0.05 for the 7.5 μg of dose of native TNF versus untreatedcontrols. § p<0.05 for the 7.5 μg of dose of the 30K-PT-cAu-TNF vectortreatment versus untreated controls and native TNF groups. *p<0.05 forthe 15 μg of dose of native or PT-cAu-TNF vector treatment versusuntreated controls and the 7.5 μg of dose native TNF. 7.5 μg of the30K-PT-cAu-TNF was not statistically different from 15 μg of native or30K-PT-cAu-TNF.

Example 12

Oral Administration of Colloidal Gold Compositions

The effect of the route of administration on the tumor sequestration ofPT-cAu-TNF vectors was tested. The vector preparation is as described inprevious Examples. Briefly colloidal gold is bound to TNF at aconcentration of 0.5 μg/ml using the in-line mixing apparatus describedabove. Following a 15-minute incubation, 30,000 MW PEG-thiol (dissolvedin pH 8 water) is added to the mixture at a final concentration of 12.5μg/ml. The solution is stirred and immediately processed bydiafiltration. The retentate is sterile filtered and aliquoted forstorage at −40° C.

MC38 tumor-burdened C57/BL/6 mice were used as a model to determine theability of orally administered PT-cAu-TNF vector to target the deliveryof TNF and gold to the tumor site. For these studies mice (n=3) wereanesthetized with 1 mg of pentobarbitol. After the animals werecompletely sedated, 26 μg of TNF bound onto the PT-cAu-TNF vector wasadministered through an oral cannula. The animals were allowed torecover and were allowed free access to food and water. The followingday, it was observed that the tumors were the familiar red/blue color ofthe colloidal gold vector. These data show oral administration of thePT-cAu-TNF vector can be used to treat tumors.

Example 13

In Vitro Activity of Colloidal Gold Bound TNF Vectors

The in vitro activity of native TNF and the cAu-TNF vectors weredetermined by the WEHI-164 TNF bioassay as described by Khabar, K. S.,Siddiqui, S., and Armstrong, J. A., WEHI-13 VAR: a stable and sensitivevariant of WEHI 164 clone 13 fibrosarcoma for tumor necrosis factorbioassay, Immunol. Lett 46: 107-110 (1995). In this bioassay, 10 ⁴WEHI-164 cells were plated in 12-well tissue culture clusters. The cellswere cultured in DMEM supplemented with 10% FBS. Native TNF and fourdifferent cAU-TNF vectors prepared at 0.5, 1, 2 and 4 ug of TNF/ml ofcolloidal gold were incubated for 7 days with the cells at final TNFconcentrations ranging from 1 mg/ml to 0.0001 mg/ml. Cell number wasdetermined on day 7 using a Coulter Counter. Data are presented as themean±SEM of the cell number for triplicate wells/TNF formulation.

The cAu-TNF vectors were biologically equivalent on a molar basis tonative TNF in the WEHI 164 bioassay. For example, 12.5 ng of native TNFinhibited WEHI 164 cell growth by 50%, whereas the same 12.5 ng dose ofthe 1.0, 2.0 and 4.0 μg/ml cAu-TNF preparations inhibited WEHI 164 cellgrowth by 47%, 55%, and 52%, respectively.

Example 14

PEG-Thiol Vector for Tumor-targeted Delivery of Antiangiogenic Drugs

These experiments used a PT-cAU-TNF-endostatin vector, a vectorcomprising two agents. It is thought that the TNF provided targetingfunctions for delivery of the therapeutic agent, endostatin (END), tothe tumor. It is also theorized that once at the target, both agents mayprovide therapeutic effects. An aspect of the vector composition is theratio of the targeting molecule, the therapeutic molecule and the PEG.All three entities are found on the same particle of colloidal gold. Aschematic of this vector is shown in FIG. 7. In FIG. 7, 1=an agent, suchas an END molecule; 2=a colloidal gold particle; 3=derivatized PEG; and4=a different agent or targeting molecule, such as a TNF molecule.

The PT-cAu_((TNF))-END vector, comprising derivatized PEG, TNF andendostatin (END) associated with a colloidal gold particle, was madeusing the apparatus described in FIG. 1. The PT-cAu_((TNF))-END was madein three steps. First, TNF associated with the gold particles at a verylow subsaturating mass of TNF. Unlike the PT-cAu-TNF vector, which wasmade with a concentration of TNF of 0.5 μg/ml, this vector was made witha TNF concentration of 0.05 μg/ml. TNF (diluted in 3 mM CAPS buffer,pH=10) which was added to the reagent bottle of the apparatus at aconcentration of 0.1 μg/ml. The second bottle in the apparatus wasfilled with an equal volume of colloidal gold at a pH of 10. TNF wasbound to the colloidal gold particles by activation of the peristalticpump as previously described. The colloidal gold-TNF solution wasincubated for 15 minutes and subsequently placed back into the goldcontainer of the apparatus. The reagent bottle was then filled with anequal volume of endostatin (diluted in CAPS buffer at a concentration of0.15 to 0.3 μg/ml. In an alternative embodiment, endostatin may bechemically modified by the addition of a sulfur group using agents suchas n-succinimidyl-S-acetylthioacetate, to aid in binding to the goldparticle.

The peristaltic pump was activated to draw the colloidal gold bound TNFand endostatin solutions into the T-connector. Upon completeinteractions of the solutions the mixture was incubated in thecollection bottle for an additional 15 minutes. The presence ofadditional binding sites for the PEG-Thiol was confirmed by the abilityof salt to precipitate the particle at this stage. After the 15 minuteincubation, 5K PEG-Thiol was added to the cAu_((TNF))-END vector andconcentrated by diafiltration as previously described.

An alternative method for binding the two proteins to the same particleof gold comprising using the same apparatus as FIG. 1 and adding theagents simultaneously to the gold. TNF and END were placed in thereagent chamber of the binding apparatus. The concentration of eachprotein was 0.25 μg/ml and as a result, 1 ml of solution contained 0.5μg of total protein. After binding the dual agent composition to goldparticles, this colloidal gold preparation also precipitated in thepresence of salt, indicating that additional free binding sites wereavailable to bind the PEG-thiol. After a 15 minute incubation, 5KPEG-Thiol was added to the cAu_((TNF))-END vector and subsequentlyprocessed as described above.

After diafiltration, the retentate was measured for TNF and ENDconcentrations in their respective EIA. To confirm the presence of ENDand TNF on the same particle of colloidal gold, a cross-antibody captureand detection assay was designed and used. A schematic representation ofthis EIA is shown in FIG. 8. In FIG. 8, A=a labeled binding partner,such as streptavidin alkaline phosphatase; B=the binding partner, suchas biotin; C=detection antibody, such as biotinylated anti-END antibody;D=an agent, such as an END molecule; F=a colloidal gold particle;E=derivatized PEG; and G=a different agent or targeting molecule, suchas a TNF molecule; H=a capture antibody; such as anti-TNF antibody; andL=a support, such as a bead or a microtiter plate.

Samples of the PT-cAu_((TNF))-END vector were added to EIA plates coatedwith either the TNF or END capturing antibodies. The samples wereincubated with the capturing antibody for 3 hours. After incubation theplates were washed and blotted dry. To bind any END present on a TNFcaptured sample, a biotinylated rabbit anti-endostatin polyclonalantibody was added to the wells. After a 30-minute incubation, theplates were washed and the presence of the biotinylated antibody wasdetected with streptavidin conjugated alkaline phosphatase. Thegeneration of a positive color signal by the endostatin detection systemindicated that the detection antibody bound to the chimeric vectorpreviously captured by the TNF monoclonal antibody. See FIG. 9. Byreversing the capturing and detection antibodies and using appropriatesecondary detection systems, an assay was used to detect the presence ofTNF on an END-captured particle. See FIG. 9. FIG. 9 is a graph showingTNF- and END-captured vectors exhibiting the presence of the secondagent.

The data from these studies are presented in Table VI. As can be seen inTable VI, the retentate of the vector samples had 17 μg/ml of TNF and 22μg/ml of END. These same samples also generated positive signals in thecross-antibody assays suggesting that both TNF and endostatin were onthe same particle of colloidal gold (FIG. 9).

TABLE VI The TNF and Endostatin concentrations present in retentates ofthe PT-cAu_((TNF))-END vector. Sample Analyte Tested ConcentrationPT-cAu_((TNF))-END TNF 17 μg/ml END 22 μg/ml

In FIG. 10 are the data showing the detection of endostatin and TNF fromthe PT-cAu_((TNF))-END vector in resected MC-38 tumors followingintravenous injection. These data show that the PT-cAu(TNF)-END vectorreached the tumor without degradation, since both molecules weredetected in the tumor tissue.

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to avector composition containing “an agent” means molar quantities of suchan agent.

It is to be understood that this invention is not limited to theparticular combinations, methods, and materials disclosed herein as suchcombinations, methods, and materials may vary somewhat. It is also to beunderstood that the terminology employed herein is used for the purposeof describing particular embodiments only and is not intended to belimiting.

1. A composition comprising for treating a solid tumor consistingessentially of a pegylated colloidal gold particle and an anti-cancertherapeutic agent, wherein a polyethylene glycol derivative iscovalently bound to the colloidal gold particle.
 2. The composition ofclaim 1, further comprising an anticancer therapeutic agent.
 3. Thecomposition of claim 2 1, wherein the anti-cancer therapeutic agentcomprises endostatin, angiostatin, thalidomide, taxol, melphalan,paclitaxel, taxanes, vinblastin, vincristine, doxorubicin, tumornecrosis factor, acyclovir, cisplatin or tacrine.
 4. The composition ofclaim 1, further comprising a targeting molecule.
 5. The composition ofclaim 1, further comprising wherein the anti-cancer therapeutic agent isbound to the colloidal gold particle by an integrating molecule.
 6. Thecomposition of claim 1, wherein the polyethylene glycol derivative is apolyethylene glycol-thiol derivative.
 7. The composition of claim 1,further comprising an immunostimulating molecule.
 8. The composition ofclaim 7, wherein the immunostimulating molecule is selected from thegroup consisting of Interleukin-1, Interleukin-2, Interleukin-3,Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7,Interleukin-8, Interleukin-10, Interleukin-11, Interleukin-12,Interleukin-13, lipid A, phospholipase A2, endotoxins, staphylococcalenterotoxin B, Type I Interferon, Type II Interferon, Tumor NecrosisFactor alpha, Transforming Growth Factor beta, Lymphotoxin, MigrationInhibition Factor, Granulocyte-Macrophage Colony-Stimulating Factor,Monocyte-Macrophage Colony-Stimulating Factor, GranulocyteColony-Stimulating Factor, Angiogenin, transforming growth factor alpha,fibroblast growth factor, cancer cell specific antigens, MART, MAGE,RAGE, B7 family of molecules and receptors, CD40 ligand/receptor, basicfibroblast growth factor, and vascular endothelial growth factor.
 9. Thecomposition of claim 1, wherein the anti-cancer therapeutic agent istumor necrosis factor alpha.